' 


BIOLOGY 

LIBRARY 

G 


r  RffiCIPLES 


OP 


JOCFEMISTRY 


T!  DENTS  OF  MEDICINE,  AGRICULTURE 
AND  RELATED  SCIENCES 


BY 


SFOR1)  ROBERTSON,  PH.D.,  D.Sc. 

It)    HIOCHEMISTRY    IN   THE    UNIVERSITY   OF   ADELAIDE,    SOUTH 
»    R  OF  BIOCHEMISTRY  IN  THE  UNIVERSITY  OF  TORONTO; 
TRY  AND  PHARMACOLOGY  IN  THE  UNIVERSITY 
OF   CALIFORNIA 


ILLUSTRATED   WITH   49    ENGRAVINGS 


LEA    &   FEBIGER 

PHILADELPHIA  AND  NEW  YORK 


\ 


LI 


DEDICATED 
TO   THE   MEMORY   OF  MY   FRIEND   AND  TEACHER 

SIR  EDWARD  C.  STIRLING 

C.M.G.,  M.A.,  M.D.,  D.Sc.  (CANTAB.),  F.R.S.,  F.R.C.S.  (ENO.) 

PROFESSOR     OF    PHYSIOLOGY  IN  THE  UNIVERSITY  OF  ADELAIDE,  SOUTH  AUSTRALIA;    DEAN  OF 

THE  FACULTY  OF  MEDICINE  AND  CONSULTING  SURGEON  OF  THE  ADELAIDE  HOSPITAL; 

CORRESPONDING    MEMBER    OF    THE    ZOOLOGICAL   SOCIETY    OF    LONDON   AND 

HONORARY    FELLOW    OF    THE    ROYAL    ANTHROPOLOGICAL    INSTITUTE 

OF   GREAT    BRITAIN   AND    IRELAND 

IN  TOKEN  OF  INTELLECTUAL  INDEBTEDNESS  AND 
PERSONAL  AFFECTION 


PREFACE. 


IT  has  been  the  object  of  the  author,  in  writing  this  book,  to  present 
the  subject  of  Biochemistry  in  close  relationship  to  Physiology,  so 
that  the  student  may  perceive  the  intimate  dependence,  of  these 
two  sciences  upon  one  another  and  come  to  regard  physiological 
chemistry  in  its  true  light,  as  the  foundation  upon  which  we  must 
ultimately  build  our  interpretations  of  the  functions  of  living  matter. 
Emphasis  has  been  placed  upon  the  practical  applications  of  the 
subject,  and  not  only  upon  applications  to  the  practice  of  medicine, 
but  also  upon  applications  to  the  industries  and  to  general  biology, 
for  while  the  design  of  the  author  has  been  primarily  to  write  a  text- 
book for  the  use  of  medical  students  and  student's  intending  to  special- 
ize in  biochemistry  and  physiology,  the  attempt  has  also  been  made 
to  compile  a  work  which  will  be  of  service  to  the  agricultural  student, 
the  student  of  general  biology,  or  the  industrial  chemist  who  is  engaged 
in  handling  biological  products. 

I  am  deeply  indebted  to  my  colleague,  Prof.  Hardolph  Wasteneys, 
for  his  valuable  cooperation  in  preparing  the  manuscript  for  the 
press,  correcting  proofs  and  compiling  the  index;  and  I  desire  to 
acknowledge  my  indebtedness  to  my  wife  for  her  assistance  in  the 
preparation  of  some  of  the  illustrations. 

T.  BRAILSFORD  ROBERTSON. 

ADELAIDE,  SOUTH  AUSTRALIA,  1920. 


CONTENTS. 


Introduction 17 

The  Nature  and  Scope  of  the  Subject 17 

The  Degree  of  Exactitude  Attainable  in  Biochemistry  ...  .21 
The  Preparation  Required  for  the  Study  of  Biochemistry  ....  24 
The  Subdivisions  of  the  Subject 27 


PART  I. 

THE  FOODS. 

• 

CHAPTER  I. 

THE  SIGNIFICANCE  OF  FOODSTUFFS. 

The  Chemical  Relationship  of  Animals  and  Plants 31 

The  Conservation  of  Matter 32 

The  Classification  of  Foodstuffs 33 

CHAPTER  II. 
THE  INORGANIC  FOODSTUFFS. 

Water  and  Sodium  Chloride .      .       34 

Calcium .      .       40 

Iron 43 

Other  Inorganic  Foodstuffs .       49 

The  Complexity  of  our  Dietary  Requirements .50 

CHAPTER  III. 
THE  CARBOHYDRATES;  THE  MONOSACCHARIDES. 

General  Characteristics 53 

The  Hexoses 55 

Reactions  of  the  Carbohydrates 58 

The  Chemical  Relationships  of  the  Sugars 63 

Certain  Derivatives  of  Glucose 66 

The  Distribution  of  the  Monosaccharides  in  Living  Tissues 69 

The  Lactone  Structure  of  Sugars 72 


CONTENTS  vii 


CHAPTER  IV. 

THE  CARBOHYDRATES;  THE  DISACCHARIDES,  POLYSACCHARIDES  AND 
GLUCOSIDES. 

The  Disaccharides 76 

Polysaccharides 81 

Aminopolysaccharides 88 

Glucosides K 89 

The  Carbohydrate  Esters     .  * . 91 

CHAPTER  V. 

THE  HYDRO-AROMATIC  DERIVATIVES:  THE  CYCLOSES,  CHOLESTEROL  AND 

CHOLIC  ACID. 

General  Characteristics  .      .      .     '; 93 

The  Cycloses 95 

Cholesterol  and  the  Phytosterols • 97 

Bile  Concretions;  Ambergris .      .      .      .  100 

Cholesterol  Esters 101 

The  Bile  Salts  and  Cholic  Acid • 102 

CHAPTER  VI. 

THE  FATS. 

The  True  Fats 107 

The  Characteristics  of  the  Natural  Fats 109 

Waxes Ill 

The  Phospholipins  or  Phosphatids 113 

Glucosides  of  the  Phospholipins 116 

CHAPTER  VII. 

THE  PROTEINS  AND  THE  AMINO-ACIDS. 

General  Characteristics  of  the  Proteins      ..........  120 

Coagulation  Reactions 122 

The  Classification  of  the  Proteins 124 

I.  The  Simple  Proteins 126 

II.  The  Conjugated  Proteins 128 

III.  The  Products  of  Protein  Hydrolysis 130 

IV.  The  Coagulated  Proteins 131 

The  End-produats  of  Protein  Hydrolysis;  the  Amino-acids        .      .      .      .      .  131 

The  Synthesis  of  Proteins 138 

The  Occurrence  of  Peptides  among  the  Products  of  Protein  Hydrolysis       .  143 
The  Analysis  and  Characterization  of  Proteins  by  the  Determination  of  the 

Amino-acid  Radicals  which  they  contain 144 

CHAPTER  VIII. 
COMPOUNDS  OF  THE  PROTEINS. 

Types  of  Union  in  the  Protein  Molecule 148 

Consequences  of  the  Polypeptide  Structure  of  Proteins 151 

The  Precipitation  and  Coagulation  of  Proteins  by  Inorganic  Salts       .      .      .  158 

Compounds  of  Proteins  with  other  Proteins 170 


viii  CONTENTS 

CHAPTER  IX. 

THE  NUCLEIC  ACIDS  AND  THE  NITROGENOUS  BASES. 

The  Decomposition-products  of  the  Nucleic  Acids    .      .      .....  .     173 

The  Structure  of  the  Nucleic  Acids -      •      •      •      •  •     178 

Amines  derived  from  Amino-acids        .      ...      •'•. -<•'.'  .      .      .      .      •  •      184 

The  Betaines  and  the  Vitamines .      .  189 

Nitrogenous  Bases  derived  from  Guanidine    .      .      .      ......      .      *  .     194 

Nitrogenous  Bases  derived  from  the  Phospholipins         196 

Nitrogenous  Bases  forming  the  Active  Principles  of  Internal  Secretions  .      197 

CHAPTER  X. 

THE  HYDROLYZING  ENZYMES. 

General  Characteristics  of  the  Enzymes 201 

The  Quantitative  Relationships  in  Hydrolysis  by  Enzymes      .      .  :    :      .  .     207 

The  Influence  of  Temperature  upon  Enzymes      .      ....      .      .      .  .     213 

The  Influence  of  Reaction  upon  Hydrolyses  by  Enzymes    .      .     ;..      .      .  .     217 

The  Specificity  of  the  Hydroly zing  Enzymes       .      .      .      ..     .      .      .      .  .     218 

The  Synthetic  Action  of  Hydroly  zing  Enzymes .      .  .221 

Antienzymes    .      .    •-,      .      .      .      .      .      .      .      .      .      ..-.'.-'".      .      .  .     226 

CHAPTER  XI. 

THE  DIGESTION  AND  ASSIMILATION  OF  THE  FOODSTUFFS. 

The  Digestion  of  the  Carbohydrates    ...      .      .      .      .      .      ^     .     .      .  .     228 

The  Digestion  of  the  Fats    .      ...... :   .,      1      ..      ...  .     232 

The  Digestion  of  the  Proteins    .      .      .      ....      ....      .      ...  .     238 

The  Time-  and  Mass-relations  of  Digestion  and  Absorption 250 


PART  II. 

THE  PROPERTIES  OF  PROTOPLASM. 


CHAPTER  XII. 

PROPERTIES  CONFERRED  BY  THE  DIFFUSIBLE  CONSTITUENTS. 

The  Osmotic  Pressure  of  the  Tissue  Fluids 255 

The  Osmotic  Pressure  of  Cell  Contents 263 

The  Composition  of  the  Mineral  Constituents  of  Tissue  Fluids      ....  268 

The  Neutrality  of  the  Tissues  and  Tissue  Fluids 271 


CONTENTS  ix 


CHAPTER  XIII. 

PROPERTIES  CONFERRED  BY  THE  COLLOIDAL  CONSTITUENTS:  STRUCTURE 
AND  CONSISTENCY. 

The  Emulsion-structure  of  Protoplasm 284 

The  Viscosity  of  Protoplasm 295 

Jellies  and  Gelatinization 298 

The  Osmotic  Pressure  of  Protein  Solutions 302 

The  Swelling  of  Protein  Jellies •'.'..  304 


CHAPTER  XIV. 

PROPERTIES  CONFERRED  BY  THE  COLLOIDAL  CONSTITUENTS:  CHEMICAL 
AND  BIOLOGICAL. 

Effects  of  Disturbance  of  the  Inorganic  Environment '  .  310 

Effects  of  Removal  of  Calcium  from  the  Tissues  and  Tissue  Fluids  .  .  .  314 
The  Mutually  Antagonistic  Action  of  Salts  and  Physiologically  Balanced 

Solutions 318 

The  Origin  of  the  Mutual  Antagonism  of  Inorganic  Salts 321 

The  Origin  of  Acid  Secretions 327 

The  Selective  Action  of  Tissues  and  the  "Oligodynamic"  Actions  of  Heavy 

Metals 328 

The  Biological  Individuality  of  Tissues  and  Tissue  Fluids 330 


PART  III. 
THE  CHEMICAL  CORRELATION  OF  THE  TISSUES. 


CHAPTER  XV. 
THE  VEHICLES  OF  CHEMICAL  CORRELATION:  BLOOD  AND  LYMPH. 

The  Composition  of  the  Blood 335 

The  Coagulation  of  the  Blood 342 

The  Chemistry  of  Hemoglobin 350 

The  Crystalline  Forms  of  Hemoglobin  in  Relation  to  the  Biological  Indi- 
viduality of  the  Blood  . 356 

The  Chemical  Detection  of  Blood .      .  361 

The  Origin  and  Composition  of  Lymph     ...........  362 


X  CONTENTS 

CHAPTER  XVI. 

EXAMPLES  OF  CHEMICAL  CORRELATION. 

The  Chemical  Correlation  of  Respiratory  Activities 365 

The  Chemical  Regulation  of  the  Circulatory  System 368 

The  Chemical  Correlation  of  the  Processes  of  Digestion       .      .      .      .      .      .371 

The  Chemical  Correlation  of  the  Organs  of  Generation 376 

The  Chemical  Regulation  of  Metabolism 381 


PART  IV. 

THE  CHEMICAL  PROCESSES  WHICH  UNDERLIE  AND 
ACCOMPANY  LIFE  PHENOMENA. 


CHAPTER  XVII. 

PROCESSES  INFERRED  FROM  DIRECT  OBSERVATION. 

The  Intermediate  Metabolism  of  the  Carbohydrates:    Muscular  Contraction  391 

The  Intermediate  Metabolism  of  the  Fats;  Diabetes      .      .      .      .      ..    ' .      .  399 

Oxidizing  Enzymes 412 

Bioluminescence 414 

CHAPTER  XVIII. 

PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION:  THE  ENERGY 
TRANSFORMATIONS  IN  LIVING  ORGANISMS. 

The  Influence  of  Temperature  upon  Life  Processes .  417 

The  Influence  of  Light  upon  Life  Processes 429 

The  Storage  of  Potential  Energy;  the  Photosynthesis  of  Carbohydrates     .  434 
The    Conversion    of    Chemical    into    Mechanical    Energy;    the    Chemical 

Mechanics  of  Muscular  Contraction 438 

CHAPTER  XIX. 

PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION:    FERTILIZATION  AND 
EARLY  DEVELOPMENT. 

The  Substitution  of  Chemical  Agencies  for  Normal  Fertilization    ....  446 

The  Nature  of  the  Agents  which  form  Fertilization  Membranes     ....  450 

The  Effect  of  Membrane-forming  Agents  upon  the  Egg 458 

The  Relationship  of  Phospholipins  to  the  Synthesis  of  Nuclear  Material  and 

the  Effects  of  Lecithin  upon  Early  Development 462 

The  Chemical  Mechanics  of  Cell  Division 466 

Artificial  Twin  Formation  and  the  Formation  of  Monstrosities  469 


CONTENTS  xi 


CHAPTER  XX. 

PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION:  GROWTH. 

General  Characteristics  of  the  Growth  Process 471 

The  Influence  of  Race,  Sex  and  Environment  upon  the  Growth  Process  .      .  484 

The  Substrates  of  Growth '.  „  ,  .      .      .      .  488 

The  Relationship  of  the  Endocrine  Organs  to  Growth    .      .      ..-.•.      .  493 

The  Metabolic  Rate  and  the  Partition  of  Nutrients       .      .      .      .'     .      .      .  500 

Catalyzers  of  Growth .      .  503 

Old  Age  and  Senescence 512 


CHAPTER  XXI. 

PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION:  MEMORY  AND  SLEEP. 

Memory 521 

The  Fatigue  Products  of  Nerve  Centers 524 

The  Application  of  the  Formula  of  Autocatalysis  to  Central  Nervous  Phe- 
nomena      527 

Sleep 530 

The  Fading  of  Memory  Traces      " 532 


PART  V. 
THE  PRODUCTS  OF  TISSUE  ACTIVITY. 

CHAPTER  XXII. 

THE  WASTE  PRODUCTS. 

The  Carbonaceous  Waste  Products      .      .      . 537 

The  Nitrogenous  Waste  Products 541 

Conjugated  Excreta 554 

Aromatic  Oxyacids 558 

Waste  Products  of  the  Sulphur  Metabolism 559 

Urinary  Pigments 562 

The  Properties  and  Composition  of  Urine 563 


CONTENTS 

PART  VI. 

THE  ENERGY-BALANCE  OF  THE  ORGANISM. 


CHAPTER  XXIII. 

THE  ANIMAL  BODY  AS  A  MACHINE. 

The  Applicability  of  the  Law  of  the  Conservation  of  Energy  to  Living 

Organisms 567 

The  Isodynamic  Values  of  the  Foodstuffs 575 

The  Protein  Requirement  in  the  Dietary .'.-.*  578 

The  Normal  Diet       .      . 582 

The  Calorific  Requirement  and  the  Surface  Law       .      .     \      .      ....  587 

The  Nutrition  of  Children    .      .      .      .      ,      ^     .     *      .      .  .  .     ,      .      .      .  590 

The  Energy  Equivalent  of  Growth       .     .  :  .      .  .;     .      .      ....      .  592 

The  Outlook   .  595 


PRINCIPLES  OF  BIOCHEMISTRY. 


INTRODUCTION. 

THE   NATURE   AND   SCOPE   OF   THE   SUBJECT. 

The  subject-matter  of  biochemistry  is  the  application  of  the  known 
principles  of  chemistry  and  physical  chemistry  to  the  study  and  inter- 
pretation of  life-phenomena;  of  the  processes,  that  is  of  digestion, 
assimilation,  respiration,  growth,  reproduction,  muscular  contraction 
and  the  like,  which  combine  to  distinguish  living  from  inanimate 
matter.  From  this  definition  it  must  be  clear  that  biochemistry 
possesses  very  close  affiliations  with  both  animal  and  vegetable  physi- 
ology. For  physiology  is  the  study  of  the  way  in  which  societies,  indi- 
viduals, organs  and  cells  perform  their  functions,  and  since  each  and 
every  function  of  living  matter  ultimately  involves  or  depends  upon 
chemical  changes,  to  this  extent  the  study  of  each  and  every  function 
of  living  matter  becomes  a  part  of  the  subject-matter  of  biochemistry. 

The  distinction  between  physiology  and  biochemistry  is  in  fact  an 
arbitrary  one,  depending  very  largely  upon  convenience  and  upon  the 
contemporary  limitations  of  our  knowledge. 

So  long  as  we  possess  no  clue  whatever  to  the  nature  of  the  processes 
which  underlie  or  accompany  a  life-phenomenon,  the  study  of  that 
phenomenon  and  of  the  method  of  its  performance  is,  beyond  any 
question,  an  exclusively  physiological  problem.  But  directly  we  take 
the  first  steps  toward  ascertaining  the  nature  of  the  chemical  phe- 
nomena which  accompany  its  performance,  we  are  taking,  also,  the 
first  steps  toward  incorporating  this  problem  into  the  subject  of 
biochemistry. 

The  historical  growth  and  development  of  the  subject  have  illustrated 
very  aptly  these  natural  applications.  In  the  beginning,  and  that  only 
one  brief  generation  ago,  biochemistry  was  an  undifferentiated  portion, 
a  minor  branch  of  physiology,  and  formed  the  subject  of  a  bare  half- 
dozen  lectures  delivered  by  the  professor  of  physiology.  Gradually 
the  need  of  special  training  for  the  study  of  this  subject,  and  its  con- 
tinually increasing  magnitude  and  practical  importance  have  led  men 
to  make  a  special  study  of  it,  apart  from  that  of  the  parent-subject. 
The  labors  of  these  men  have  quickly  added  countless  phenomena 
to  their  special  domain,  and  so  important  are  these,  and  so  funda- 
mental is  the  part  which  biochemistry  now  plays  in  medicine,  agri- 
2 


18  PRINCIPLES-  OF  BIOCHEMISTRY 

culture  and  the  industries,  that  almost  everywhere  the  study  of  bio- 
chemistry ranks  with  that  of  anatomy,  physiology  and  pathology  as 
one  of  the  studies  fundamental  to  the  understanding  of  medical  science, 
or  with  botany,  plant-physiology  and  bacteriology  as  one  of  the  studies 
fundamental  to  the  understanding  of  agriculture. 

It  must  not  be  supposed,  however,  that  the  withdrawal  of  biochem- 
istry from  the  parent-subject  has  left  physiology  any  the  poorer. 
Physiology  has  not  been  left  merely  with  a  residuum  of  undigested 
material,  ultimately  to  be  absorbed  by  the  biological  chemist.  On 
the  contrary,  with  the  development  of  biochemistry,  physiology  has 
developed  too  and  that  to  an  extent  unimagined  by  its  founders.  A 
few  generations  ago,  physiology  was  a  little-considered  fragment  of  the 
study  of  anatomy,  just  as,  one  generation  ago,  biochemistry  was  a 
little-considered  portion  of  the  study  of  physiology.  The  same  differ- 
entiation has  separated  the  teaching  of  biochemistry  from  physiology 
as  that  which  has  separated  the  teaching  of  chemistry  from  that 
of  physics.  We  may  regard  physiology  as  consisting  for  the  present 
of  the  study  of  the  applications  of  anatomy  and  physics  to  the  elucida- 
tion of  life-phenomena,  together  with  the  entire  study  of  a  residuum  of 
phenomena  and  processes  which  are  for  the  present  passed  by  in 
biochemistry  simply  because  we  do  not  as  yet  possess  any  clue  whatever 
to  the  nature  of  the  chemical  processes  which  underlie  them. 

Hence,  physiology  is  destined  ultimately  and  at  some  as  yet  far 
distant  date  to  become  the  study  of  the  interpretation  of  life-phenomena 
by  the  aid  of  the  principles  of  anatomy,  gross  and  minute,  and  physics. 
Biochemistry  is  the  study  of  the  interpretation  of  life-phenomena  by 
the  aid  of  the  principles  and  facts  of  chemistry.  Physiology  investi- 
gates the  molar  and  molecular  phenomena  of  life,  biochemistry  the 
atomic. 

Of  course,  this  division  is  arbitrary  and  unreal,  just  as  the  distinc- 
tion between  physics  and  chemistry  is  arbitrary  and  unreal.  Nature 
recognizes  no  such  classification  of  her  phenomena.  Physics  merges 
insensibly  into  chemistry  and  in  like  manner  physiology  merges  into 
biochemistry.  An  illustration  of  this  fact  has  been  strikingly  afforded 
in  recent  times  by  the  rapid  development  of  physical  chemistry,  a 
whole  borderland  between  physics  and  chemistry,  which  has  undergone 
such  extensive  survey  within  the  last  generation  as  to  demand  a 
noteworthy  degree  of  special  training  on  the  part  of  those  who  would 
•attempt  to  master  it.  Even  the  delineation  of  this  domain  has  not  by 
any  means  removed  all  of  the  "debatable  land,"  however,  that  lies 
between  physics  and  chemistry;  witness  the  recently  discovered 
phenomena  of  radio-activity,  which  have  opened  up  yet  another  field 
of  investigation  which  is  neither  physics  nor  chemistry.  And  so  it  is 
with  physiology  and  biochemistry.  There  is  an  indefinite  "debatable 
area"  between  the  two,  and  many  if  not  most  of  the  problems  in  either 
field  require  the  aid  of  both  physiology  and  biochemistry  for  their 
solution. 


NATURE  AND  SCOPE  OF  THE  SUBJECT  19 

The  investigator  of  Nature  cannot  afford  to  hamper  himself  by 
arbitrary  definitions  and  delimitations  of  his  field.  When  the  need 
arises  he  must  be  prepared  to  use  the  tools  which  the  problem  calls 
for,  be  they  the  tools  of  physics,  chemistry,  mathematics,  anatomy, 
bacteriology  or  pathology.  The  teacher  is  somewhat  more  con- 
strained. He  cannot  carry  his  pupils  too  far  from  the  center  of  the 
subject  in  hand,  lest  their  lack  of  preparation  should  render  them 
unable  to  follow  him.  Even  so,  however,  the  student  of  biochemistry 
will  often  have  occasion  to  dwell,  in  his  studies,  upon  certain  aspects  of 
problems  which  the  physiologist  has  made  peculiarly  his  own,  and  the 
medical  student  will  frequently  find  himself  studying  one  and  the  same 
problem  in  his  course  in  biochemistry  and  again  in  his  course  in 
physiology.  Nevertheless  he  will  find  that  he  is  not  merely  repeating 
his  work,  not  merely  covering  old  ground,  but  that,  on  the  contrary,  the 
physiologist  and  the  biochemist  have  each  of  them  something  different 
to  say;  displaying  the  problem  in  different  lights  and  dwelling  upon  it 
in  different  connections. 

We  have  stated  that  the  science  of  biochemistry  consists  in  the 
interpretation  of  life-phenomena  in  the  light  of  the  facts  and  principles 
of  chemistry.  The  question  may  here  very  naturally  arise  in  the  mind 
of  the  reader,  How  can  it  be  possible  to  apply  chemistry  to  the  investi- 
gation of  living  matter?  True,  we  can  attempt  to  analyze  living  mat- 
ter, to  separate  chemical  constituents  from  it  and  to  identify  them. 
But  then,  directly  we  begin  to  analyze  living  matter  it  ceases  to  be 
living  matter.  The  reagents  which  we  employ  immediately  "kill" 
it,  that  is  to  say,  abruptly  suspend  its  characteristic  functions  and 
disperse  and  dissolve  the  minute  structures  of  protoplasm  which  are  the 
physical  substratum  upon  which  its  functional  activities  are  reared. 
Unquestionably,  an  amoeba  which  has  been  boiled  in  hydrochloric 
acid  may  yield  interesting  products,  but  then  it  is  no  longer  an  amoeba, 
and  the  products  which  analysis  yields  bear  only  a  remote  relationship 
to  those  which  were  originally  present  in  the  living  organism. 

To  find  out  what  is  actually  occurring  in  living  matter-  we  must, 
therefore,  employ  methods  of  investigation  somewhat  analogous  to 
those  which  the  physical  chemist  employs  in  the  investigation  of  what 
is  actually  occurring  in  flames.  First,  we  study  the  nature  of  the 
substances  which  enter  the  flame,  then  we  study  the  properties  and 
behavior  of  the  flame  itself,  always  taking  care  to  do  so  by  the  aid  of 
instruments  which  do  not  disturb  the  flame,  and  finally  we  ascertain 
what  substances  the  flame  gives  off.  From  these  various  and  frag- 
mentary data  we  endeavor  to  reconstruct  in  our  minds  a  coherent 
picture  of  the  train  of  events  as  they  actually  occur,  and  this  endeavor 
will  be  the  more  successful  in  proportion  to  the  extent,  the  variety  and 
exactitude  of  our  measurements. 

So  far  as  possible,  then,  we  must  bring  static  and  not  dynamic  methods 
of  mensuration  to.  the  study  of  living  matter,  methods,  that  is,  which  do 
not  involve  the  cessation  of  the  very  processes  which  we  desire  to 


20  PRINCIPLES  OF  BIOCHEMISTRY 

investigate.  Often,  it  is  true,  we  can  successfully  employ  destructive, 
dynamic  methods  to  find  out  many  important  things.  For  example, 
in  the  study  of  digestion,  we  can  destroy  the  living  cells  which  form  the 
lining  mucous  membrane  of  the  stomach,  and,  having  destroyed  them, 
extract  from  them  a  substance,  Pepsin,  which  will  digest  proteins,  even 
in  glass  vessels  in  laboratory-incubators.  In  this  way  and  by  similar 
methods  we  can  study  the  changes  which  are  brought  about  in  our 
foodstuffs  when  they  enter  the  alimentary  canal.  Even  in  a  fairly 
simple  case  such  as  this,  however,  the  dynamic  method  does  not 
altogether  suffice.  For  we  find  that  within  the  alimentary  canal  itself 
the  foods  are  digested  much  more  rapidly  than  we  can  digest  them  with 
the  aid  of  ferments  in  laboratory-glassware.  Some  condition,  other 
than  mere  warmth  or  mechanical  agitation,  some  condition  which  we 
have  not  yet  fully  succeeded  in  imitating,  very  materially  aids  the 
action  of  these  ferments  in  the  cavity  of  the  living  alimentary 
canal. 

By  such  phenomena  as  these  we  are  constantly  being  reminded  that 
it  is  not  by  any  means  safe  to  argue  directly  from  the  behavior  of  dead 
fragments  or  products  of  living  tissue,  to  that  of  living  tissue  itself. 
The  results  of  dynamic  experiments  which  involve  the  actual  destruction 
of  the  living  tissues  which  we  are  investigating,  only  afford  a  starting- 
point,  therefore,  or  an  orientation,  for  our  guidance  in  a  repetition  of  the 
experiment  under  actual  living-conditions. 

Biochemistry,  therefore,  falls  very  naturally  into  two  fields  of  study, 
differentiated  by  the  methods  of  investigation  employed.  The  one 
field,  that  which  has  until  recently  been  the  peculiar  interest  of  the 
"physiological  chemists,"  consists  in  the  study  of  the  crude  substances 
which  enter  into  the  life-flame  and  the  products  which  leave  it.  The 
foodstuffs  and  the  excreta,  and,  incidentally,  the  composition  of  dead 
matter  that  once  was  living,  also  the  study  of  the  action  and  reaction 
of  fragments  of  living  or  dead  protoplasm  upon  the  foods  or  upon  one 
another,  these,  until  comparatively  recently,  comprised  the  whole 
activity  and  interest  of  chemistry  in  the  investigation  of  living  matter. 
It  is  obvious,  however,  that  while  knowledge  of  these  things  is  an 
essential  prerequisite  to  the  understanding  of  the  chemical  phenomena 
of  life,  yet  they  are  far  from  yielding  information  as  to  the  nature  of 
life-processes  themselves.  It  was  for  this  reason,  and  with  justice, 
that  one  of  the  greatest  contributors  to  our  knowledge  in  this  field, 
G.  von  Bunge,  exclaimed  in  1894,  "All  processes  in  the  organism  which 
may  be  explained  mechanically  are  no  more  phenomena  of  life  than  are 
the  movements  of  the  leaves  and  branches  of  a  tree  that  is  shaken  by 
the  storm,  or  the  movement  of  the  pollen  that  the  wind  wafts  from  the 
male  poplar  to  the  female."1  We  were  at  that  time  hovering  upon  the 
outskirts  of  the  main  problems,  since  actual  penetration  of  them  was 
necessarily  deferred  until  the  momentous  advances  of  physical  chemis- 

1  Lchrbuch  der  Physiologischen  und  Pathologischou  Chcraio,  3tc  Aufl.,  Leipzig,  1894. 


EXACTITUDE  ATTAINABLE  IN  BIOCHEMISTRY  21 

try  placed  in  our  hands  the  necessary  implements  and  knowledge  to 
essay  the  task. 

Our  second  field  of  study,  then,  consists  in  an  analysis  of  the  chemical 
phenomena  which  accompany  or  underlie  the  activities  of  living,  undis- 
turbed, and  more  or  less  normally  functioning  protoplasm,  a  field  which 
until  recently  was  almost  exclusively  the  preoccupation  of  the  "  experi- 
mental biologist."  Inevitably,  however,  these  two  phases  of  chemical 
inquiry,  so  closely  affiliated,  so  mutually  dependent,  are  coming  to  rely 
more  and  more  intimately  upon  each  other  and  hence  are  being  welded 
more  and  more  firmly  into  one.  Experimental  biology  drawing  upon 
the  rich  resources  of  physiological  chemistry,  is  immensely  increasing 
its  exactitude  and  its  certainty,  while  physiological  chemistry,  on  the 
other  hand,  is  rapidly  widening  the  horizon  of  its  inquiries  in  response 
to  inspiration  drawn  from  the  field  of  experimental  biology.  In  this 
work  we  will  recognize  no  distinction  between  these  fields,  but  endeavor, 
in  so  far  as  the  limitations  of  our  knowledge  permit,  to  interweave  them 
into  one  coherent  representation  of  the  complex  tissue  of  chemical 
processes  which  constitutes  life  and  its  immediate  consequences. 

THE  DEGREE  OF  EXACTITUDE  ATTAINABLE  IN  BIOCHEMISTRY. 

In  the  so-called  "exact  sciences,"  to  wit,  mechanics  and  physics, 
we  have,  as  a  rule,  the  power  to  isolate  more  or  less  completely  any 
phenomenon  or  group  of  phenomena  which  we  wish  to  study,  and  to 
guard  them  from  disturbance  by  the  intrusion  of  accidental  variables. 
For  example,  it  is  not  a  difficult  matter  to  demonstrate  that  a  falling 
body  experiences  a  constant  acceleration,  the  most  serious  intrusive 
variable  being  the  friction  of  the  air,  a  variable  which  can  now  be  very 
readily  excluded  in  a  variety  of  ways.1  Similarly,  in  chemistry,  it  is 
not  a  difficult  matter  to  observe  the  progress  and  equilibrium  of  such 
a  reaction,  as,  for  example,  the  reduction  of  iron  oxide  by  hydrogen. 
The  chemicals  are  procurable  in  pure  conditions,  only  one  reaction 
occurs,  and  it  is  a  simple  matter  to  exclude  other  chemicals  and  to  keep 
the  temperature  and  pressure  of  the  system  constant.  In  organic 
chemistry  much  more  complex  phenomena  are  encountered.  It  is 
the  exception  rather  than  the  rule  to  find  a  reaction  which  proceeds 
evenly  and  without  disturbance  by  side-reactions  or  secondary  decom- 
positions. To  detect  regularities  and  establish  "laws"  hi  such  a  system 
is  a  task  the  more  complex  the  greater  the  number  of  adventitious 
variables. 

The  difficulties  which  are  encountered  in  studying  organic  reactions 
in  laboratory  glassware  are  enormously  magnified  in  studying  reactions 

1  It  must  be  remembered  that  the  friction  of  the  air,  which  to  us  presents  no  difficulty, 
was  to  our  ancestors  an  insuperable  obstacle  to  the  measurement  of  gravity.  In  exactly 
the  same  way  insurmountable  obstacles  which  at  this  day  defeat  our  ends  in  physiologi- 
cal or  biochemical  research  will  appear  of  trivial  importance  to  our  intellectual  heirs. 
As  a  rule  such  obstacles  merely  imply  that  we  are  attacking  the  problem  from  the  wrong 
angle. 


22  PRINCIPLES  OF  BIOCHEMISTRY 

which  occur  in  living  matter.  The  life  of  any  one  cell  consists  in  a 
multiplicity  of  parallel  reactions,  interrelated,  interdependent,  and 
interwoven  into  a  bewildering  complex.  Multicellular  organisms,  such 
as  ourselves,  consist  of  millions  of  such  cells.  When  the  reader  is 
reminded  that  the  reactions  in  each  organ  or  group  of  cells  and  possibly, 
even  in  each  individual  cell,  possess  an  individual  character  of  their  own, 
and  that  these  reactions  are  excessively  sensitive  to  external  agencies, 
the  complexity  of  the  task  of  unravelling  the  separate  reactions  and 
tracing  their  individual  progress  must  be  evident. 

It  follows  from  the  complexity  of  the  phenomena  that  the  regularities 
and  relations  observed  by  the  biochemist  are  rarely  capable  of  formula- 
tion with  such  precision  as  those  which  are  observed  by  the  physicist 
or  chemist.  To  illustrate  this  fact,  let  us  consider  the  difficulties 
attendant  upon  the  investigation  of  one  of  our  simplest  problems, 
to  wit,  that  of  the  mode  of  action  of  the  protein-digesting  ferment 
Trypsin.  We  have  first  of  all  to  overcome  the  difficulty  of  obtaining 
pure  protein.  That  obtained  (and  a  "pure"  protein  in  the  sense  that 
inorganic  reagents  may  be  "pure"  has  never  been  prepared),  we  then 
have  the  difficulty  of  obtaining  a  pure  trypsin,  a  difficulty  which  has 
never  been  even  partially  overcome.  In  fact  we  certainly  possess  no 
pure  trypsin  and  we  have,  moreover,  no  method  of  ascertaining  how 
impure  our  preparations  are.  Not  only  are  our  preparations  of  trypsin 
impure,  but  they  frequently  contain  several  ferments  which  digest 
proteins,  a  fact  which  has  only  recently  come  to  be  appreciated. 
Notwithstanding  all  these  obstacles  we  have  found  that  if  trypsin 
be  allowed  to  act  upon  protein,  with  certain  necessary  precautions,  a 
regularity  may  be  observed  in  the  rate  of  decomposition  of  the  protein 
by  the  ferment,  and  this  regularity  may  even  be  formulated  in  mathe- 
matical terms.  We  are  not  surprised  to  find,  however,  that  the  agree- 
ment between  the  formula  and  the  experimental  measurements  (of 
quantity  of  protein  digested)  is  not  extremely  exact.  Under  very 
favorable  conditions  the  requirements  of  theory  and  the  findings  of  the 
investigator  may  agree  to  within  one  per  cent,  of  their  mean  value. 
In  a  purely  chemical  problem  an  agreement  to  within  one-tenth  of  a 
per  cent,  is  anticipated  and  not  infrequently  obtained.  In  physics 
or  in  astronomy  an  agreement  to  within  one  one-hundredth  of  a  per 
cent,  is  not  in  the  least  exceptional.  As  the  uncontrollable  adventitious 
variables  become  fewer,  it  will  be  observed,  the  agreement  between 
formulae  and  experimental  data  becomes  more  and  more  precise. 

Hypotheses  of  a  more  general  character,  not  admitting  of  mathe- 
matical formulation,  share  in  this  disadvantage,  and  hence  it  arises 
that  a  larger  proportion  of  hypotheses  in  biological  sciences  are  of 
uncertain  or  very  questionable  validity  than  of  those  in  the  so-called 
"exact"  sciences.  But  the  difference  is  merely  a  matter  of  degree 
and  tends  progressively  to  diminish.  All  scientific  hypotheses  and 
"laws"  are  subject  to  a  marginal  inexactitude,  and  all  human  precision 
is  relative.  As  our  acquaintance  with  any  field  of  investigation  grows 


EXACTITUDE  ATTAINABLE  IN  BIOCHEMISTRY  23 

more  extensive,  the  width  of  the  margin  of  inexactitude  diminishes,  and 
that  is  all.  For  example,  no  "law"  is  apparently  of  more  extensive 
applicability  or  capable  of  more  precise  mathematical  formulation 
than  Newton's  law,  that  bodies  attract  one  another  as  the  inverse 
square  of  their  distance  apart.  Yet  certain  astronomical  data,  devia- 
tions in  the  orbit  of  the  planet  Mercury,1  point  to  the  possibility  that 
even  this  law  may  not  be  exact  and  that  the  true  exponent  of  the 
distance  may  in  truth  not  be  2  but  2.000,0001612.  The  margin  of 
inexactitude  is  here  represented  by  the  minute  fraction  0.0000001612, 
but  it  is  here  nevertheless.  And  so  it  is  with  all  scientific  hypotheses. 
Our  laws,  formulations  and  hypotheses  are  merely  temporary  short- 
hand statements  of  our  acquaintance  with  the  facts.  As  our  acquaint- 
ance with  the  facts  grows  larger  we  must  revise  our  shorthand  to  express 
our  accessions  of  knowledge.  The  shorthand  is  not  the  knowledge 
itself.  Science,  in  reality,  consists  solely  in  our  knowledge  of  facts  and 
our  control  of  the  forces  of  nature  and  not  of  the  hypotheses  which  we 
formulate  by  the  way  in  order  to  summarize  our  present  state  of 
knowledge  and  stimulate  the  imagination  to  fresh  inquiries.  Biology 
is,  in  truth,  no  less  an  "exact"  science  than  any  other,  than  astronomy, 
for  example,  but  its  hypotheses  are  subject  to  much  more  frequent 
and  thorough  revision  than  those  of  physics  or  astronomy,  simply 
because  our  knowledge  of  the  field  is  less  and  is  growing  more  rapidly. 

The  whole  theory  of  the  scientific  method  of  thought  has  in  fact 
been  based  by  the  great  founders  of  science  upon  the  assumption  of  the 
fallibility  of  purely  intellectual  operations,  and  hence  of  the  untrust- 
worthiness  of  hypotheses.  Newton's  famous  rule,  "  Hypotheses  non 
fingo,"  while  impracticable  for  the  individual  investigator,  remains 
nevertheless  true  of  science  as  a  whole,  of  the  body  of  exact  knowledge, 
that  is,  which  endures  the  test  of  time  and  endows  mankind  with  the 
power  of  ruling  and  directing  the  multifarious  and  stupendous  forces  of 
Nature.  During  the  centuries  which  have  been  marked  by  the  acquire- 
ment of  this  knowledge  countless  hypotheses  have  been  formed,  and 
accepted  for  a  while,  and  then  abandoned  as  evidently  absurd.  But 
the  forward  march  of  exact  knowledge  has  never  suffered  interruption 
and  not  infrequently  indeed  has  been  very  much  facilitated  by  the 
most  obviously  erroneous  hypotheses.  The  phlogiston  theory  of  heat 
is  perhaps  the  most  striking  example  of  this  kind.  It  was  a  most 
patently  erroneous  hypothesis,  built  up  by  perfectly  sound  reasoning 
based  upon  imperfectly  understood  facts.  Yet  for  a  hundred  years 
the  mere  existence  of  this  hypothesis  was  the  greatest  contemporary 
stimulus  to  the  development  of  chemistry  and  it  ultimately  led  to  the 
establishment  of  the  conception  of  the  conservation  of  matter. 

As  the  curves  of  the  geometrician  approach  and  yet  never  actually 
attain  their  asymptote,  so  do  we  continually  approach  and  never  yet 
have  we  attained  the  utter  truth.  The  merit  of  the  scientific  method 

1  Cf .  article  on  Gravitation,  Encyclopedia  Britannica,  1 1th  edition. 


24  PRINCIPLES  OF  BIOCHEMISTRY 

of  thought  lies  in  the  fact  that  the  otherwise  circular  speculations  of 
humanity,  ever  returning  unprofitably  to  the  point  from  which  they 
started,  have  had  a  thrust  communicated  to  them  which  has  deflected 
them  into  a  perpetually  widening  spiral,  reaching  further  and  ever 
further  into  the  infinite,  promising  knowledge  commensurate  only  with 
the  immensity  of  the  universe,  and  power  to  which  no  man  dare  set  a 
limit. 

If,  then,  our  present  conceptions  in  biochemistry  are  subject  to  rapid 
and  comprehensive  modification  this  affords  no  legitimate  basis  for 
scientific  cynicism  or  indiscriminate  scepticism.  On  the  contrary  it  is 
a  hopeful  augury,  testifying  to  the  youth  of  the  subject  and  the  vast 
development  that  lies  before  it.  No  subject,  indeed,  promises  more 
immediate  developments  of  stupendous  significance  to  man.  The 
control  of  life  itself,  no  less,  is  the  alluring  aim  and  destiny  of  the 
medical  and  biological  sciences  and  the  basis  of  every  step  in  the 
acquirement  of  this  control  must  inevitably  be  founded  on  a  knowledge 
of  the  chemical  processes  which  underlie  and  constitute  life.  We  may 
be  well  content,  with  such  a  prospect  before  us,  to  resign  absolute 
certainty  to  the  political  doctrinaire.  For  ourselves,  dwelling  amid 
uncertainties  and  hazards,  advancing  like  bold  navigators  in  uncharted 
seas,  we  will  turn  our  faces  toward  the  new  and  wider  horizons  which 
always  lie  before  us.  We  will  regard  a  hypothesis  as  an  instrument  of 
research,  like  a  balance,  a  burette,  or  better  still  a  compass;  a  guide 
and  a  stimulus  to  investigations,  but  a  mere  approximation  to  the  truth 
which  we  trust  will  gradually  approach  closer  and  yet  closer  to  verity 
as  our  knowledge  grows  in  extent  and  proliferates  in  detail. 

THE  PREPARATION   REQUIRED   FOR   THE   STUDY   OF 
BIOCHEMISTRY. 

No  amount  of  courage  and  enthusiasm,  however,  will  suffice  to  alto- 
gether compensate  for  lack  of  preliminary  training  and  acquired  skill 
in  those  branches  of  science  upon  which  biochemistry  is  founded  and 
from  which  it  originates.  Biochemistry  is  in  the  first  place  and  most 
essentially  an  outgrowth  from  organic  chemistry  and  an  acquaintance 
with  the  general  principles  of  that  science  and  the  simpler  laboratory 
procedures  most  frequently  employed  in  it,  is  as  essential  to  the 
understanding  of  biochemistry  as  a  vocabulary  of  French  words  is  to 
the  understanding  of  Moliere  in  the  original.  In  this  work  I  will 
suppose  the  reader  to  be  acquainted  with  organic  structural  formulas 
and  the  general  principles  according  to  which  they  are- inferred  from  the 
behavior  of  the  substances  to  which  they  are  applied.1 

The  modern  developments  of  biochemistry  and  particularly  those 
which  aim  at  the  interpretation  of  the  processes  underlying  the  per- 
formance of  function,  involve  the  application  of  the  elementary  princi- 

1  The  reader  whose  previous  training  in  this  subject  has  been  dencient  may  consult 
E.  V.  McCollum,  Organic  Chemistry  for  Students  of  Medicine,  New  York,  1916. 


PREPARATION  FOR  THE  STUDY  OF  BIOCHEMISTRY        25 

pies  of  physical  chemistry  and  there  can  be  ho  doubt  whatever  that  the 
future  and  most  momentous  developments  of  the  subject  are  destined 
to  involve  physical  chemistry  more  and  more  extensively.  The  essen- 
tial principles  are  neither  numerous  nor  abstruse,  good  elementary 
text-books  of  the  subject  abound  and  the  student  is  earnestly  recom- 
mended, if  he  has  not  previously  received  training  in  this  subject,  to 
acquire  for  himself  a  suitable  handbook  of  physical  chemistry,1  and  to 
consult  it  frequently  in  the  course  of  his  studies  in  biochemistry. 

The  intelligent  employment  of  the  elementary  principles  of  physical 
chemistry  implies  a  nodding  acquaintance  with  the  so-called  "higher 
mathematics,"  but  far  more  than  for  the  mere  understanding  of 
physical  chemistry,  mathematics  is  an  essential  instrument  in  the 
handling  of  quantitative  measurements  of  any  kind  whatsoever. 
Every  branch  of  science  is,  in  its  youth,  qualitative,  and  in  its  maturity 
quantitative.  Even  taxonomy  has  been  converted  by  the  discoveries  of 
Mendel  into  a  quantitative  study  involving  in  some  instances  very 
complex  mathematical  operations.  Biochemistry  is  at  the  present 
juncture  passing  through  a  species  of  adolescence  and  emerging  by  very 
rapid  stages  from  the  qualitative  into  the  quantitative  stage  of  develop- 
ment. The  student  who  would  prepare  himself  for  the  future,  there- 
fore, would  do  well  to  acquire  such  rudiments  of  mathematical  skill 
as  may  be  necessary  for  the  elucidation  of  principles  which .  he  will 
unquestionably  be  called  upon  to  comprehend.  Mathematics  is  in 
reality  a  symbolic  language  which  expresses  in  brief  terms  a  series  of 
interrelated  facts  and  considerations  which  would  otherwise  be  too 
intolerably  complex  to  retain  simultaneously  in  the  mind.  By  acquir- 
ing mathematical  facility,  therefore,  the  student  is  not  augmenting 
the  complexity  of  his  task,  but  simplifying  it. 

The  applications  of  physical  chemistry  to  biological  problems 
necessitate  of  course  an  elementary  knowledge  of  the  differential 
calculus  and  the  simplest  methods  of  integration.2  All  of  the  work 
upon  ferments  and  digestion  in  its  quantitative  and  most  important 
aspects  now  demands  the  employment  of  the  calculus.  As  an  example 
of  the  wider  and  at  first  sight  unexpected  applications  of  this  mathe- 
matical technique  the  reader  is  referred  to  the  important  work  of  Barcroft,3 
which  has  marked  an  epoch  in  our  understanding  of  the  respiratory 
functions  of  the  blood  and  which  could  never  have  yielded  one  tithe 
of  the  information  obtained  without  the  employment  of  the  methods  of 
the  differential  and  integral  calculus. 

A  moderate  familiarity  with  the  elementary  principles  involved  in 
the  solution  of  differential  equations  would  also  upon  occasion  be  found 

1  For  example,   E.  W.  Washburn:     An  Introduction   to  the  Principles  of    Physical 
Chemistry,  New  York,  1915.   A.  Findlay:    Practical  Physical  Chemistry,  London,  1914. 

2  The  student  may  consult  J.  Edwards:      Differential  Calculus  for  Beginners    and 
Integral  Calculus  for  Beginners,  while  for  the  methods  of  applying  the   calculus  to  the 
solution  of   scientific  problems  the  student  would  do  well  to  read  Perry's   Calculus  for 
Engineers,  London,  1897. 

3  The  Respiratory  Function  of  the  Blood,  Cambridge,  1914. 


26  PRINCIPLES  OF  BIOCHEMISTRY 

very  useful.1  In  the  treatment  of  quantitative  data  and  the  graphic 
representations  thereof  it  is  frequently  necessary  or  desirable  to  apply 
a  formula  to  the  curves  obtained  or  to  compare  them  with  the  curve 
which  may  be  deduced  from  theoretical  premises.  In  this  extensive 
field  of  practice  a  knowledge  of  the  proper  method  of  dealing  with  and 
minimizing  the  effect  of  accidental  experimental  errors  is  required  and 
the  employment  of  the  method  of  least  squares  is  essential  if  the  best 
use  is  to  be  made  of  the  experimental  material  which  may  be  available.2 
For  purposes  of  fitting  empirical  formulae  to  curves,  eliminating  ex- 
cessively erroneous  results  and  interpolating  probable  values  between 
values  which  have  actually  been  measured,  a  study  of  the  methods  of 
interpolation  and  mechanical  differentiation  is  exceedingly  valuable 
and  helpful.3 

But  perhaps  the  most  essential  branch  of  mathematical  practice  in 
the  equipment  of  the  biochemist  of  the  future  will  consist  in  the 
methods  of  the  statistician.  When  we  come  to  deal  with  actually 
living  material,  as  we  are  compelled  to  do  in  order  to  advance  our 
subject  at  all  in  its  most  significant  direction,  we  are  at  once  con- 
fronted by  the  problem  created  by  the  inherent  variability  of  living 
things.  No  two  animals  are  alike,  not  even  may  we  find  any  two  living 
cells  which  are  precisely  identical.  In  agriculture  no  two  plots  of 
ground  are  alike,  no  two  plants  are  ever  identical.  How  then,  in 
comparing  experimental  animals  or  plants  or  plots  of  ground  with 
"normals"  or  "controls"  shall  we  ever  attain  to  certainty  of  our 
results?  It  would  seem  that  it  must  always  be  possible  that  the 
differences  between  any  two  groups  of  animals  may  merely  be  the  pro- 
duct of  chance  selection  of  two  groups  which  might  have  differed  in  the 
observed  sense  without  any  experimental  manipulation  whatsoever. 
This  difficulty,  the  fundamental  character  of  which  is  recognized  by 
every  biological  investigator,  is  of  course  not  of  so  much  importance 
in  those  cases  in  which  the  differences  for  which  we  are  looking  are  very 
large,  as  death  contrasted  with  survival,  decisive  loss  of  weight  con- 
trasted with  equally  decisive  gain,  or  reduction  or  enhancement  of 
normal  qualities  by  fifty  per  cent,  or  more.  But  phenomena  such  as 
these  are  the  obvious  ones  in  any  field  of  science,  those  which  lie  at  the 
surface  and  are  garnered  by  the  earliest  investigators,  and  they  are  not 
invariably,  and  in  fact  not  usually,  the  phenomena  upon  which  we 
ultimately  come  to  rely  for  the  basis  of  wide  and  fundamental  generaliza- 
tions. Such  emphatic  disparities  testify  in  themselves  to  the  unusual- 
ness  of  the  conditions  invoked,  and  hence  carry  the  suspicion  that  the 
response  to  such  extreme  conditions  may  not  be  a  normal  or  at  least  a 
usual  reaction  of  living  matter  to  its  environment.  For  our  deeper 

1  Murray:     Introductory  Course  in  Differential  Equations,  London,   1897. 

2  M.  Merriman:     Text-book  on  the  Method  of  Least  Squares,  New  York,  1891.     L. 
Tuttle:     The  Theory  of  Measurement,  Philadelphia,   1916. 

8  H.  L.  Rice:  The  Theory  and  Practice  of  Interpolation,  Lynn,  Mass.,  1899. 
J.  Mellor:  Higher  Mathematics  for  Students  of  Chemistry  and  Physics,  London,  1902. 


PREPARATION  FOR   THE  STUDY  OF  BIOCHEMISTRY        27 

understanding  of  life  which  is  to  come  therefore,  we  must  learn  to 
rely  with  confidence  upon  relatively  small  and  fluctuating  disparities 
between  groups  composed  of  very  variable  material.  This  can  be 
done  in  one  way  and  in  one  way  only,  namely,  by  employing  the  methods 
of  the  statistician  whereby  we  may  accurately  gauge  the  relative 
values  of  observations  obtained  with  variable  material,  compute  the 
number  of  observations  necessary  to  attain  a  given  degree  of  certainty 
or  accuracy,  place  in  their  proper  perspective  extreme  or  overlapping 
variations  in  aberrant  individuals  and,  in  short,  render  measurements 
upon  even  such  variable  material  as  living  organisms  just  as  precise 
as  the  measurements  employed  in  quantitative  analysis. 

The  student  of  biochemistry  would  be  well-advised  therefore  to 
acquire  the  simple  mathematical  technique  which  is  requisite  for  the 
employment  of  statistical  methods,1  but  he  should  remember  that  this 
branch  of  mathematics  above  all  others  abounds  in  pitfalls  for  the 
unwary  and  he  should  be  sure  that  he  perfectly  comprehends  the 
simple  fundamental  principles  which  underlie  these  methods  before  he 
attempts  to  put  them  into  practice.2  If  the  reader  should  desire  to 
gain  a  conception  of  the  variety  and  scope  of  the  possible  applications 
of  the  statistical  method  to  problems  of  biochemistry,  experimental 
biology  and  agriculture,  he  may  consult  the  recent  work  of  Loeb  and 
Wasteneys  upon  the  applicability  of  the  Bunsen-Roscoe  law  to  animal 
heliotropism,3  of  Waynick  upon  the  distribution  of  nitrifying  bacteria 
in  soils4  and  of  the  author  upon  the  growth  of  children.5 

The  Subdivisions  of  the  Subject. — In  this  work  we  will  endeavor 
to  follow  up  the  foodstuffs  from  the  moment  when  they  are  partaken 
of,  to  the  moment  when,  after  having  circulated  through  the  body  and 
partaken  of  its  life,  their  final  products  are  excreted.  The  subject- 
matter  is  divided  into  six  parts  corresponding  with  various  phases  of 
the  cycle  of  changes  which  the  foodstuffs  undergo.  The  subdivisions 
are  as  follows: 

Part  I. — The  Foods,  their  properties,  digestion,  assimilation  and 
conversion  into  living  matter  or  into  reserve-materials.  The  considera- 
tion of  this  phase  of  our  subject  takes  us  up  to  the  point  at  which  the 
foodstuffs,  subjected  to  certain  modifications,  have  really  been  converted 
into  living  protoplasm.  This  leads  us  naturally  to  the  consideration 
of  the  second  phase  of  our  subject,  namely : 

1  The  Student  may  consult  G.  Udney  Yule:      An  Introduction  to  the  Theory  of 
Statistics,  London,    1911.      For  tables  and  formulae  the  student  may  refer  to  C.  B. 
Davenport:     Statistical  Methods,  New  York,   1904. 

2  Probably  the   best   introduction    to   the   fundamental   conceptions   of   probability 
which  form  the  basis  of  the  statistical  method  is  contained  in  the  classical  little  memoir 
of  W.  A.  Whitworth  entitled  Choice  and  Chance,  Cambridge,   1901. 

3  Jour.  Exper.  Zool.,  1917,  22,  187. 

4  D.   D.  Waynick:     University  of  California  Publications  in    Agricultural  Sciences, 
1918,  3,  243. 

*  T.  Brailsford  Robertson:  Am.  Jour.  Physiol.,  1915,  37,  1  and  74;  1916,  41,  535, 
and  547. 


28  PRINCIPLES  OF  BIOCHEMISTRY 

Part  II. — The  manner  in  which  the  properties  of  the  foodstuffs 
mould  and  determine  the  properties  of  living  protoplasm. 

Part  III. — In  proceeding  to  consider  the  activities,  apart  from  the 
merely  passive  properties  of  living  matter,  we  are  at  once  confronted 
with  the  significant  fact  that  the  multicellular  organisms,  like  our- 
selves, are  really  immense  societies  composed  of  innumerable  minute 
units  which  are  the  individual  living  cells.  We  have,  in  this  society, 
a  governing  authority,  the  central  nervous  system;  a  postal-telegraphic 
system,  the  peripheral  nervous  system;  a  laboring  class,  the  muscles 
and  glandular  tissue-cells;  distributing  agencies,  the  blood  and  lymph, 
and  with  all  of  these  not  a  rigid  central  autocratic  control,  but  a  very 
considerable  degree  of  local  autonomy.  Every  cell  is  working,  not  by 
deliberate  instruction,  but  as  a  part  of  its  very  specialized  life.  In 
order  to  avoid  confusion  in  so  vast  a  complex  of  semi-independent 
units,  numerous  cooperative  mechanisms  must  be  present  to  adjust 
supply  to  demand  and  effort  to  need.  These  mechanisms  imply  a 
certain  correlation  of  distant  .parts;  for  instance,  between  the  neuro- 
muscular  system  which  controls  the  respiratory  movements,  and  the 
need  of  the  tissues  for  oxygen.  This  correlation  of  different  and  often 
widely  separated  activities  is  brought  about  by  the  interaction  of  two 
distinct  types  of  agency,  nervous  agencies  and  chemical  agencies.  In 
so  far  as  this  correlation  of  activities  is  brought  about  by  chemical 
means,  it  will  fall  under  consideration  in  this  third  phase  of  our  subject. 

Part  IV. — In  this  part  we  will  endeavor  to  attack  the  very  kernel  of 
our  problem,  that  part  of  our  studies  which  is  destined  to  provide  the 
ultimate  foundation  of  the  practice  of  medicine  and  in  no  small  measure 
of  agriculture  also.  The  chemical  phenomena  which  underlie,  accom- 
pany or  even  actually  constitute  the  living  activities  of  cells  will  here 
be  our  preoccupation  and  we  will  incidentally  study,  so  far  as  our 
fragmentary  knowledge  at  this  time  permits,  the  changes  which  the 
foodstuffs  or  constituents  of  protoplasm  undergo  at  the  instant  of  their 
utilization  for  the  furtherance  of  vital  functions.  Here  we  will  find 
our  most  alluring  problems  and  our  least  extensive  knowledge,  here  is 
the  region  in  which  must  occur  the  greatest  conquests  which  lie  before 
us  and  those  which  will  exercise  the  most  fundamental  and  far-reaching 
effect  upon  our  own  lives  and  the  lives  of  those  who  will  follow  after  us. 

Part  V. — In  this  part  we  will  take  up  the  study  of  the  waste-products 
which  ultimately  result  from  the  activities  of  oiir  tissues;  the  ashes,  the 
products  of  combustion  and  the  debris  which  result  from  the  daily 
maintenance  and  furtherance  of  life. 

Part  VI. — In  this  part,  regarding  the  entire  body  as  a  chemical 
machine,  somewhat  crudely  comparable  to  a  steam-engine,  we  will 
discuss  the  question  of  the  efficiency  of  the  machine  and  the  relation- 
ship of  the  horse-power  it  can  develop  to  the  nature  and  value  of  the 
fuel  with  which  it  is  provided.  It  is  in  this  connection  that  we  will 
discuss  data  which  may  enable  us  in  some  measure  to  answer  the 
question,  what  investment  of  particular  types  and  mixtures  of  fuel  will 


PREPARATION  FOR  THE  STUDY  OF  BIOCHEMISTRY        29 

return  the  greatest  interest  in  the  form  of  efficient  work  on  the  part  of 
this  very  complex  machine,  a  human  being?  (Given,  of  course  the 
incalculable  psychological  asset  of  good-will.)  This  is  the  type  of 
problem  with  which  the  allied  governments  and  Germany  were  recently 
grappling  and  in  proportion  as  we  can  contribute  to  its  answer  we 
are  assisting  not  merely  to  guide  civilization  and  humanity  safely 
through  the  most  dangerous  crisis  of  all  its  long  history,  but  also  to 
solve  a  perennial  problem  which  the  War  merely  rendered  acute  a 
little  earlier  than  would  otherwise  have  been  the  case,  the  problem, 
namely,  of  correlating  production  and  distribution  with  the  fluctuating 
needs  of  the  scattered  populations  of  the  world.  The  specialization 
of  the  occupations  of  peoples  and  areas  which  has  so  characterized 
the  development  of  civilization  in  the  past  century  carries  with  it 
inherent  dangers  which  approach  more  and  more  near  as  the  process 
of  specialization  extends.  The  specialized  individual  is  always  depen- 
dent upon  others  for  his  support.  The  specialized  city  or  nation  is 
dependent  upon  the  world.  The  mutual  dependency  of  peoples  which 
our  multifarious  modern  activities  has  evoked  compels  attention,  in 
widely  separated  parts  of  the  world,  to  the  needs  of  remote  and  alien 
workers.  These  needs  are  chemical  in  their  basis  and  biochemistry 
alone  can  supply  us  with  the  exact  knowledge  which  is  necessary  to 
adjust  them. 


PART   I. 

THE  FOODS 


CHAPTER   I. 
THE  SIGNIFICANCE  OF  FOODSTUFFS. 

THE  CHEMICAL  RELATIONSHIP   OF   ANIMALS  AND  PLANTS. 

In  considering  the  nature  of  the  foods  and  their  elaboration  into 
living  matter,  it  is  necessary  in  the  first  place  to  realize  that  the  foods 
of  multicellular  animals  such  as  ourselves,  are,  at  the  same  time,  the 
constituents  out  of  which  living  matter  is  built  up.  This  becomes 
evident  when  we  recollect  that  the  majority  of  our  foodstuffs  consists 
of  matter  that  was  formerly  living  or  which  is  derived  from  matter 
that  was  formerly  living.  Meats  and  vegetables  and  grains  are,  of 
course,  matter  that  wras  awhile  ago  alive,  that  is  now  arrested  in  its 
function  and  more  or  less  rapidly  decomposing  into  more  elementary 
substances,  but  still  contains,  for  the  most  part,  the  components  of 
living  protoplasm.  Perhaps  they  are  not  linked  together  in  precisely 
the  way  in  which  they  are  linked  together  in  truly  living  matter,  and 
perhaps  the  fact  that  this  matter  is  no  longer  living  is  attributable  to 
this  disturbance  in  the  linkage  of  its  constituents.  Still  the  con- 
stituents are  there,  and  we  appropriate  them,  modify  them  in  some 
degree,  and  build  them  up  into  our  own  tissues.  Other  foodstuffs, 
such  as  sugar,  are  directly  extracted  from  living  tissues  in  which  they 
form  stores  or  reserves  of  energy,  for  example  from  beets  or  sugar-canes, 
and  these  are  likewise  appropriated  to  our  own  use. 

In  this  respect  we  differ  very  materially  from  the  plants,  the  foods 
of  which  are  in  general  very  much  more  elementary  than  ours.  Plants 
are  actually  able  to  build  up  living  tissues  out  of  substances  which  have 
in  themselves  no  necessary  connection  with  living  protoplasm;  out  of 
mineral  salts,  water  and  carbon  dioxide.  For  this  reason  it  used  to  be 
thought  that  only  plants  possessed  the  powrer  of  synthesizing  the  actual 
constituents  of  living  matter  and  that  we,  without  doing  any  fresh 
construction,  simply  sort  out  and  appropriate  these  preformed  con- 
stituents and  thus  live  in  a  state  of  parasitism  upon  the  vegetable 
world. 


32        ,-,«;.»t..    .SIQtflFIQANCE  OF  FOODSTUFFS 

The  discovery  by  Schmiedeberg  and  Bunge  in  1876  of  the  synthesis 
of  hippuric  acid  from  benzoic  acid  and  glycocoll  in  the  tissues  of  the 
kidney,1  disposed  of  this  untenable  distinction,  and  while  we  are  cer- 
tainly to  be  regarded  as  primarily  parasitic  upon  the  vegetable  and 
lower  orders  of  the  animal  kingdom,  yet  we  are  not  so  unable  to  create 
constituents  of  living  matter,  as  earlier  investigators  imagined.  We 
know  now  that  animal  tissues  can  perform  a  multiplicity  of  syntheses 
whereby  constituents  of  protoplasm  are  made  which  the  food  does  not 
contain  preformed.  It  will  be  found,  however,  to  be  a  general  charac- 
teristic of  syntheses  carried  out  in  animal  tissues,  that  the  storage  of 
energy  or  heat-value  which  is  accomplished  thereby  is  usually  small, 
whereas  in  green  plants  syntheses  are  accomplished  which  involve  the 
locking  up,  for  longer  or  shorter  periods,  of  very  large  quantities  of 
energy;  for  eons  as  in  coal  deposits,  or  for  the  brief  period  of  a  single 
winter  as  in  the  seeds  which  consume  their  stored-up  energy  when 
they  germinate  in  the  spring. 

The  reason  for  this  distinction  is  not  far  to  seek.  The  green  plant 
has  an  inexhaustible  reservoir  of  energy  upon  which  to  draw;  the 
radiant  energy  of  the  sun;  and  the  energy  which  is  locked  up  in  the 
starches,  fats  and  proteins,  which  plants  synthesize  from  the  most 
elementary  products  of  combustion,  is  derived  in  the  long  run  from  the 
sun.  The  animal  has  no  comparable  capital  to  draw  upon,  and  if  an 
animal  is  to  perform  a  synthesis  involving  absorption  of  heat  or  energy, 
it  can  only  do  so  at  the  expense  of  its  current  account,  that  is  to  say 
by  the  degradation  of  its  own  tissues  or  food  reserves.  On  the  whole, 
therefore,  and  with  the  exceptions  noted,  green  plants  are  the  prime 
conservers  of  energy,  while  the  function  of  animals  is  to  dissipate  it 
again.  The  whole  fever  and  bustle  of  life  upon  the  earth  is  therefore 
none  other  than  a  transitory  phase  through  which  continually  passes 
a  minute  fraction  of  the  colossal  outpourings  of  solar  energy. 

THE   CONSERVATION   OF  MATTER. 

Whatever  may  be  the  relative  efficiency  of  different  types  of  proto- 
plasm as  storers  of  energy  and  creators  of  living  matter,  they  are  all 
alike  subject  to  the  law  of  the  Conservation  of  Matter.2  That  is  to  say, 
although  an  animal  or  plant  cell  may  create  new  chemical  compounds ; 
new  permutations  and  combinations  of  the  chemical  elements,  it  cannot 
create  new  elements.  All  of  the  carbon  in  its  tissues  must  have  been 
derived,  for  example,  from  carbon  from  without.  If  an  animal  gives 
off  nitrogen  in  the  form  of  urea,  it  must  either  take  up  fresh  nitrogen 
from  without,  or  else  its  tissues  must  remain  permanently  poorer  in 
nitrogen. 

1  Thai  this  synthesis  occurs  in  some  organ  or  tissue  of  the  body   had  been  recognized 
by  Wohler  as  early  as  1824. 

2  The  applicability  of  the  law  of  the  Conservation  of  Matter  to  living  organisms  was 
first  demonstrated  by  its  discoverer,  the  French  chemist  Lavoisier    (1743-1794),  and 
subsequently  confirmed  in  detail  by  Licbig  (1803-1873). 


CLASSIFICATION  OF  FOODSTUFFS  33 

Now  it  is  self-evident  that  we  are  continually  voiding  waste  products, 
urea  and  very  many  other  substances  in  the  urine,  carbon  dioxide  and 
water-vapor  in  the  breath,  and  various  items  of  waste  in  the  sweat 
and  in  the  feces.  Furthermore,  despite  his  rapid  and  continual  losst 
of  substance,  when  we  are  adult  we  remain  tolerably  constant  in  weight 
and  composition,  that  is,  if  we  are  healthy  and  neither  becoming 
emaciated  nor  growing  fat.  It  follows  that,  as  a  general  rule,  we  must 
be  taking  in  from  without,  not  only  just  as  much  total  substance  as  we 
are  losing  daily  in  these  various  ways,  but  also  just  as  much  of  each  of 
the  individual  elements,  nitrogen,  carbon  and  so  forth,  as  we  are  daily 
voiding.  This  intake  of  elements  constitutes  the  act  of  feeding,  and 
the  forms  in  which  we  take  in  these  elements  are  our  Foods. 

THE   CLASSIFICATION   OF   FOODSTUFFS. 

As  has  been  stated,  our  articles  of  diet  are  more  complex  than  those 
of  the  plants.  Plants  can  utilize  the  carbon  in  carbon  dioxide,  but  we, 
in  order  to  replace  our  carbon-waste,  must  use  some  more  complex 
compound  of  carbon,  in  fact,  as  our  daily  experience  reveals,  either  a 
carbohydrate  (sugars,  starches,  etc.),  a  fat,  or  a  protein.  Otherwise 
we  inevitably  suffer  from  carbon  starvation.  Plants,  again,  can  derive 
nitrogen  from  nitrates  in  the  soil,  but  we,  more  dependent,  can  only 
derive  the  nitrogen  which  we  need  from  preformed  protein.  Mineral 
and  other  inorganic  foods  we  only  utilize  to  replace  or  provide  mineral 
or  inorganic  constituents  of  our  tissues ;  we  cannot  utilize  them  directly 
to  build  up  carbohydrates  or  fats  as  plants  can.  Our  foodstuffs  fall, 
therefore,  into  four  main  classes,  to  wit : 

1.  The  Inorganic  Foods,  such  as  water  and  mineral  salts. 

2.  The  Carbohydrates,  such  as  the  sugars  and  starches. 

3.  The  Fats. 

4.  The  Proteins. 

To  which  must  be  added  certain  accessory  articles  of  diet,  to  which 
frequent  reference  will  be  made,  which  are  of  vital  importance  to  the 
maintenance  and  furtherance  of  life,  but  yet  do  not  necessarily  fall 
within  any  of  the  above-mentioned  classes. 


CHAPTER   II. 
THE  INORGANIC  FOODSTUFFS. 

WATER  AND   SODIUM  CHLORIDE. 

We  can  readily  understand  how  the  need  for  the  organic  foodstuffs 
arises:  the  fats,  carbohydrates  and  proteins.  For  they  are  fuels  which 
in  the  course  of  combustion  give  up  a  certain  number  of  heat-units 
which  can  be  utilized  in  the  performance  of  all  the  work  which  an 
animal  daily  accomplishes.  We  can  also  readily  understand  how  the 
need  for  Water  arises.  Protoplasm  consists  very  largely  of  water. 
Over  70  per  cent,  of  our  body-weight  is  water  and  consequently  we 
living  animals  are  reservoirs  or  sacks  of  water  which  are  at  the  same 
time  porous.  Just  as  an  earthenware  jar  containing  water  will  gradu- 
ally but  continuously  lose  the  water  by  evaporation  from  the  outer 
surface  of  the  jar,  so  we  also  lose  water  continually,  by  evaporation 
from  the  skin  and  from  the  respiratory  epithelium  in  the  lungs,  apart 
from  the  water  which  is  daily  lost  in  urine  and  which  serves  the  purpose 
of  flushing  the  excreta  out  of  the  conduits  of  the  body.  Consequently 
a  need  for  water  arises,  a  need  of  the  cells  and  tissues  which  is  expressed 
in  our  consciousness  by  that  indefinite  sensation  which  we  call  "  thirst." 

But  it  is  not  so  clear  why  we  should  require  Mineral  Salts.  \Ve  do 
not  decompose  them.  They  can  yield  us  no  energy.  It  is  not  at  once 
evident  why  we  should  lose  them  as  we  cannot  help  losing  water.  Yet 
we  do  lose  them  daily  and  that  daily  loss  must  be  replaced.  We  daily 
take  in  sodium  chloride  and  it  reappears  as  sodium  chloride  in  the 
urine.  At  the  end  of  its  passage  through  the  tissues  it  appears  unal- 
tered, yet  it  has  unquestionably  performed  a  function  and  indeed  many 
functions  during  its  sojourn  in  our  bodies. 

The  probable  nature  of  some  of  these  functions  will  be  more  clearly 
apprehended  at  a  later  stage,  when  we  take  up  the  consideration  of  the 
relationship  between  the  properties  of  living  matter  and  those  of  its 
constituents.  But  having  regard  at  present  only  to  the  beginning  and 
the  end  of  •  the  cycle  of  processes  in  which  the  mineral  salts  of  the  diet 
take  part  as  they  pass  through  the  body,  the  question  presents  itself: 
what  is  the  daily  loss  of  mineral  salts  and  what  must  be  the  daily  intake 
to  recoup  the  body  for  this  loss? 

The  mineral  a^lts  which  are  found  in  our  tissues  are,  for  the  most 
part,  supplied  in  abundance  in  our  diet.  We  do  not  consciously  seek 
for  them  as  desirable  in  themselves.  A  remarkable  exception  to  this 
rule  is  afforded  by  common  salt,  sodium  chloride,  of  which  we  feel 


WATER  AND  SODIUM   CHLORIDE  35 

impelled  to  seek  an  additional  supply.  This  fact  is  the  more  remark- 
able because  all  of  our  ordinary  articles  of  food  contain  abundance  of 
sodium  chloride,  yet  however  much  of  other  diet  we  may  eat  we  still 
experience  salt-hunger,  a  hunger  which  under  certain  conditions,  may 
become  positively  distressing. 

In  this  connection  it  is  noteworthy  that  a  very  close  parallelism 
exists  between  the  nature  of  the  diet  of  different  animals  and  peoples 
and  their  requirement  of  salt;  a  parallelism  which  was  first  pointed  out 
and  interpreted  by  the  physiological  chemist  von  Bunge. 

Very  many  of  the  animals  whose  diet  is  purely  vegetarian  experience 
a  desire  for  salt.  Carnivorous  animals,  on  the  contrary,  such  as  the 
dog  or  cat,  not  only  do  not  desire  salt,  but  actually  exhibit  an  aversion 
for  salted  food.  This  is  very  well  illustrated  by  well-known  habits  of 
many  of  the  wild  animals.  It  is  a  fact  commented  upon  in  nearly  every 
book  of  traveller's  and  hunter's  tales,  that  the  hoofed  animals,  the  deer 
and  so  forth,  of  which  the  dietary  is  exclusively  vegetable,  deliberately 
seek  for  salt,  in  salt-pools  and  efflorescences,  where  they  lick  the  salt, 
and  will  travel  very  long  distances  to  do  so.  As  all  readers  of  travel 
and  adventure  know,  it  is  at  salt  licks  that  hunters  watch  for  such 
game.  On  the  other  hand,  salt  has  never  any  attraction  for  the  wild 
beasts  of  prey. 

This  difference  of  behavior  becomes  all  the  more  striking  when  we 
reflect  upon  the  fact  that,  weight  for  weight,  a  herbivorous  animal 
takes  in  with  its  ordinary  food  just  about  the  same  quantity  of  sodium 
and  chlorine  per  day  as  a  carnivorous  animal.  Each  receives  the  same 
allowance  of  salt.  Yet  the  herbivora  experience  a  longing  for  more 
salt  and  the  carnivora  do  not. 

The  reason  is  obviously  to  be  sought  in  some  other  difference  between 
their  contents  of  sodium  chloride.  Now  one  very  striking  difference 
is  found  between  the  mineral  contents  of  vegetable  and  animal  food. 
Vegetables  nearly  all  contain  a  superabundance  of  potassium  salts. 
Animal  flesh,  on  the  contrary,  contains  sodium  and  potassium  in  nearly 
equal  proportions,  so  that  although  a  herbivorous  animal  obtains  just 
as  much  sodium  chloride  per  day  as  a  carnivorous  animal,  .yet  it 
obtains  in  many  cases  no  less  than  six  times  as  much  potassium  as  a 
carnivorous  animal  does.  It  is  in  fact  a  general  rule,  to  which  there  are 
but  few  exceptions,  that  in  plant-tissues  potassium  predominates  very 
greatly  over  sodium,  while  in  animal  tissues  these  mineral  bases  are 
present  in  approximately  equal  proportion. 

Von  Bunge  sought  to  trace  the  origin  of  the  craving  which  herbiv- 
orous animals  experience  for  salt  to  the  excess  of  potassium  in  the  diet. 
Suppose  that  a  salt  of  potassium,  say  potassium  citrate,  gains  entrance 
into  the  blood  by  having  been  ingested  with  the  food.  On  arriving  in 
the  blood-stream,  the  potassium  citrate  meets  with  an  excess  of  sodium 
chloride,  for  in  the  blood-plasma,  or  fluid  part  of  the  blood,  sodium 
predominates  very  greatly  over  potassium.  Of  course  a  certain  degree 
of  interchange  of  ions  will  take  place.  A  proportion  of  the  potassium 


36  INORGANIC  FOODSTUFFS 

citrate  will  react  with  sodium  chloride  to  form  potassium  chloride  and 
sodium  citrate,  in  accordance  with  the  equation: 

CHzCOOK  CH2COONa 

I /OH  I  /OH 

C  +     3NaCl        '     C  +     3KC1 


I  \COOK 


\COONa 


CHzCOOK  CH2COONa 

and  an  excess  of  sodium  citrate  appears  in  the  blood  together  with  an 
unusual  excess  of  potassium  chloride.  Of  course  a  similar  interchange 
would  take  place,  with  analogous  results,  if  the  salt  ingested  were 
potassium  tartrate,  malate  or  any  other  of  the  organic  salts  of  potas- 
sium which  are  so  frequently  abundant  in  vegetable  tissues. 

Now  it  is  a  function  of  the  kidneys,  as  the  reader  will  soon  come  to 
appreciate  very  fully,  to  keep  the  composition  of  the  blood  very 
nearly  constant.  They  act,  in  fact,  with  the  utmost  precision,  picking 
out  and  rejecting  abnormal  or  excessive  constituents.  The  composition 
of  the  blood  cannot  vary  beyond  the  slightest  extent  without  the 
supervention  of  grave  disturbances  involving  all  the  tissues  of  the  body. 
As  a  result  of  the  ingestion  of  potassium  citrate,  tartrate,  malate  or 
other  potassium  salts  which  are  found  in  vegetables,  we  have  seen  that 
a  new  salt  of  sodium  is  formed  in  the  blood-plasma,  to  wit,  sodium 
citrate,  tartrate,  malate  or  what  not.  This  abnormal  constituent  is 
straightway  picked  out  and  eliminated  by  the  kidneys,  together  with 
as  much  as  possible  of  the  excess  of  potassium  chloride,  and  thus  as  a 
result  of  the  ingestion  of  potassium  salts  the  blood  is  robbed  of  both 
sodium  and  chlorine. 

This  theoretical  deduction  can  very  readily  be  illustrated  experi- 
mentally. Von  Bunge  collected  his  urine  from  day  to  day  and 
measured  the  diurnal  excretion  of  sodium.  He  then  simply  added 
18  grammes  of  K2O,  in  the  form  of  citrate  or  phosphate,  to  his  daily 
diet.  The  twenty-four-hour  excretion  of  sodium  (estimated  as  Na2O) 
immediately  increased  by  8  grammes.  Now  18  grammes  of  K2O  is 
not  at  all  an  unusual  amount  to  ingest  along  with  a  vegetable  diet. 
If  one  were  to  satisfy  one's  protein  requirements  with  potatoes,  as 
many  Irish  peasants  do,  for  example,  one  would  obtain  no  less  than 
40  grammes  of  K2O  per  diem. 

One  result  of  subsisting  upon  a  vegetable  diet,  therefore,  is  a  con- 
tinual abstraction  of  sodium  and  chlorine  from  the  blood.  Now  the 
blood  resists  most  strongly  any  alteration  in  its  composition.  The 
reader  will  come  to  appreciate  more  and  more  clearly  as  this  work 
progresses,  how  intimately  the  most  fundamental  activities  of  the  body 
are  dependent  for  their  continuance  upon  the  unalterable  composition 
of  the  blood.  The  slightest  alteration  even  in  the  ratio  of  sodium  to 
potassium  in  the  blood  would  work  havoc  with  our  tissue-activities. 
Hence  the  blood  must  recoup  itself,  and  it  can  only  recoup  itself  by 


WATER  AND  SODIUM  CHLORIDE  37 

abstracting  sodium  and  chlorine  from  the  tissues.  Hence  the  tissues, 
in  consequence  of  a  vegetable  diet,  are  robbed  of  sodium  chloride. 
They  experience  salt-hunger,  a  want  which  finds  psychological  expres- 
sion in  an  indefinable  longing  for  things  which  taste  salt. 

That  the  desire  for  salt  which  so  many  herbivorous  animals  experi- 
ence is  really  attributable  to  the  nature  of  their  diet  is  remarkably 
illustrated  by  the  habits  of  various  human  races.  Von  Bunge  has 
collected  together  by  exhaustive  inquiries  from  travellers,  explorers, 
and  works  of  travel,  a  quantity  of  information  regarding  the  consump- 
tion of  salt  among  different  peoples.  Only  to  cite  a  few  among  very 
numerous  instances:  Country  people,  in  Europe  at  all  events  where 
habits  have  become  fixed  by  centuries  of  adherence  to  the  soil,  eat 
more  vegetables  and  less  animal  food  than  the  dwellers  in  cities.  For 
instance  in  France,  where  the  collection  of  internal  revenue  upon  salt 
facilitates  the  acquirement  of  statistical  data,  it  has  been  found  that 
the  consumption  of  salt  per  head  is  three  times  as  great  in  the  country 
districts  as  in  the  cities.  Then  there  are  whole  tribes  of  nomads  in 
various  parts  of  the  world  who  are  hunters,  such  as  certain  tribes  of 
the  old  North  American  Indians,  some  Arabian  and  Siberian  tribes 
and  the  Bushmen  of  South  Africa.  These  people  live,  or  used  to  live 
exclusively  upon  a  flesh-diet  and  they  never  taste  salt.  In  fact,  as  a 
rule,  they  find  salt  very  disagreeable  and  consider  the  use  of  it  by 
Europeans  ridiculous.  Not  only  is  this  the  case  with  tribes  who  have 
lived  for  generations  upon  a  flesh-diet,  but  it  applies  also  to  Europeans 
who  visit  them  and  adopt  their  diet.  Thus  one  traveller  informed 
von  Bunge  that  while  he  lived  among  the  Tunguses,  an  exclusively 
carnivorous  tribe  which  dwells  in  Siberia,  he  lived  entirely  upon 
reindeer-flesh  and  game,  and  never  experienced  the  slightest  desire 
for  salt  or  inconvenience  from  the  lack  of  it. 

Very  different  was  the  experience  of  the  Scotch  explorer,  Mungo 
Park,  when  travelling  among  the  negro  tribes  of  West  Africa.  These 
people  live  upon  a  mixed  diet  containing  a  very  high  proportion  of 
vegetables.  Salt  is  very  rare  in  their  country,  and,  as  the  vegetable 
diet  causes  a  longing  for  salt,  Park  states  that  among  the  natives,  to 
say  that  a  man  eats  salt  with  his  meals  was  equivalent  to  saying  that 
he  was  rich.  In  Park's  own  words:  "In  the  districts  of  the  interior 
salt  is  the  greatest  of  all  delicacies.  It  strikes  a  European  very  strangely 
to  observe  a  child  sucking  a  piece  of  rock-salt  as  if  it  were  sugar.  I 
have  frequently  seen  this  done.  I  myself  have  found  the  scarcity  of 
this  natural  product  very  trying.  Constant  vegetable  food  causes  a 
painful  longing  for  salt  that  is  quite  indescribable.  On  the  coast  of 
Sierra  Leone  the  desire  for  salt  is  so  keen  among  the  negroes  that  they 
gave  away  wives,  children,  and  everything  that  was  dear  to  them,  in 
return  for  it." 

Hunting  tribes,  therefore,  who  subsist  on  flesh,  experience  no  need 
for  salt  and  never  eat  it  even  when  it  is  easy  to  obtain.  Agricultural 
tribes,  on  the  contrary,  experience  a  keen  desire  for  salt.  A  peculiar 


38  INORGANIC  FOODSTUFFS 

confirmation  of  von  Bunge's  interpretation  of  this  phenomenon  is 
afforded  by  the  custom  of  one  tribe  to  which  von  Bunge  refers,  the 
negro  inhabitants  of  a  region  in  the  neighborhood  of  Khartoum. 
These  natives  manufacture  or  formerly  manufactured  a  salt  of  their 
own,  by  igniting  the  ash  of  a  plant  belonging  to  Salsola  or  salt-wort 
group.  As  has  been  stated,  the  majority  of  plants  contain  a  much 
larger  proportion  of  potassium  than  of  sodium.  The  plants  of  the 
Salsola  group  are  quite  peculiar  in  the  respect  that  their  ash  contains 
a  much  higher  proportion  of  sodium  than  of  potassium.  The  employ- 
ment of  this  particular  plant-ash  among  all  the  others  that  might 
have  been  tried  can  hardly  be  considered  accidental,  in  other  words 
it  must  have  been  found  to  satisfy  a  desire  not  equally  readily  satisfied 
by  the  ashes  of  other  plants. 

The  relation  of  a  need  for  salt  to  the  partaking  of  a  vegetable  diet 
has  had  several  peculiar  historical  consequences.  For  example,  in  the 
Mosaic  Law,  the  Jews  are  expressly  commanded  to  present  their 
vegetable  offerings  to  the  Deity  accompanied  by  salt.  In  Greek  and 
Roman  times,  sacrificial  animals  were  offered  up  to  the  Gods  without 
salt,  but  the  fruits  of  the  earth  with  salt. 

The  effect  of  eating  salt  with  our  food  is  therefore,  to  widen  the 
circle  of  palatable  foods,  We  all  know  how  insipid  potatoes  taste 
without  salt.  That  is  probably  attributable  to  their  high  content  of 
potassium,  unusual  even  in  plants.  By  adding  salt  to  our  diet  we  are 
able  to  render  potatoes  palatable,  and  so  with  many  more  foodstuffs 
of  vegetable  origin. 

So  far,  all  of  the  facts  which  we  have  cited  are  in  excellent  harmony 
with  the  view  that  a  diet  containing  an  excess  of  potassium  salts  gives 
rise  to  a  necessity  and  a  desire  for  common  salt.  Not 'every  animal 
appears  to  experience  this  desire,  however,  for  rabbits  and  hares,  for 
example,  live  on  a  diet  containing  an  excess  of  potassium  salts  and  yet 
do  not  seek  for  salt  and  do  not  appear  to  experience  any  inconvenience 
from  lack  of  it.  Domestic  herbivorous  animals  will  live  without  incon- 
venience on  a  purely  vegetable  diet  without  salt  indefinitely,  although 
they  will  eat  salt  when  it  is  offered  them  and  unmistakably  find  it 
gratifying.  None  of  these  live  on  a  diet  so  excessively  rich  in  potassium 
as  potatoes,  for  example,  but  nevertheless  there  is  no  question  but 
that  they  must  ingest  a  large  excess  of  potassium  salts.  Yet  the 
blood-plasma  of  such  animals  remains  of  the  usual  composition, 
containing  an  excess  of  sodium  over  potassium. 

Here  we  meet  for  the  first  time  with  a  phenomenon  which  is  of  very 
general  occurrence  in  living  matter,  namely  the  phenomenon  of  selec- 
tive assimilation  by  tissues.  Living  tissues,  as  we  shall  have  occasion 
to  note  many  times,  are  not  mere  passive  recipients  of  whatever  may 
be  contained  in  the  fluids  which  bathe  them.  They  choose  and  select 
suitable  ingredients  in  suitable  proportions  and  reject  unsuitable  or 
excessive  ingredients.  A  remarkable  illustration  of  this  is  afforded  by 
an  experiment  of  Landsteiner's.  He  fed  young  rabbits  upon  meadow 


WATER  AND  SODIUM  CHLORIDE  39 

hay  exclusively  for  three  and  a  half  months.  At  the  same  time  a 
similar  batch  of  animals  was  fed  exclusively  upon  cow's  milk.  Now 
these  two  diets  contained  very  different  relative  amounts  of  sodium 
and  potassium,  hay  being  much  richer  in  potassium  than  in  sodium, 
and  milk  richer  in  sodium  than  in  potassium.  Yet  at  the  end  of  the 
period  the  composition  of  the  blood  obtained  from  the  two  groups, 
as  regards  sodium  and  potassium,  was  identical.  The  tissues,  not  only 
the  epithelium  of  the  kidney  but  that  of  the  intestine  as  well,  actively 
choose  the  constituents  which  they  will  reject  or  absorb  respectively. 
In  just  the  same  way  a  plant,  living  in  water  rich  in  sodium  and  poor 
in  potassium,  will  nevertheless  pick  the  potassium  out  and  build  it 
up  into  tissues  which  are  rich  in  potassium  and  poor  in  sodium.  But 
this  power  of  selection  is  limited,  and  in  extreme  cases,  as,  for  example, 
a  diet  so  rich  in  potassium  as  potatoes,  some  aid  is  required,  and  sodium 
and  chlorine  in  the  form  of  common  salt  must  be  added  to  the  dietary. 

From  the  standpoint  of  physical  chemistry  it  is  of  course  evident 
that  selective  absorption  of  mineral  salts  by  the  epithelium  of  the 
intestine  or  their  selective  elimination  by  the  kidneys  must  involve 
the  performance  of  work;  the  expenditure  of  energy.  For  the  osmotic 
pressures  of  the  various  salts  in  the  solutions  bathing  the  cells  would 
tend  to  drive  them  into  the  absorbing  or  excreting  tissues  in  pro- 
portion to  their  concentration  and  if,  on  the  contrary,  they  appear  on 
the  other  side  of  these  epithelial  tissues  in  emphatic  disproportion  to 
their  original  concentrations,  the  process  of  assimilation  or  excretion 
must  have  involved  the  overcoming  of  the  forces  of  Osmotic  Pressure. 
The  energy  necessary  to  achieve  this  can  only  be  derived  from  the 
combustion  of  other  foodstuffs  or  constituents  of  tissues  which  are  thus 
robbed  of  the  supplies  available  for  carrying  on  the  other  activities 
of  the  body.  Selective  absorption  or  excretion  implies  work,  therefore, 
and  anything  which  relieves  the  tissues  in  any  measure  of  the  necessity 
of  exercising  selection  sets  free  a  certain  number  of  heat-units  for  other 
uses  or,  in  other  words,  improves  the  utilization  of  other  foodstuffs. 
The  gratification  and  frequent  improvement  in  nutrition  which 
accompanies  the  administration  of  salt  to  herbivorous  animals  may 
thus  originate  in  relief  of  the  tissues  from  the  strain  and  burden  of 
selection  and  the  liberation  of  foodstuffs  for  the  maintenance  of  other 
tissue-activities  which  is  in  effect,  equivalent  to  the  addition  of  a  certain 
amount  of  food  to  the  accustomed  dietary.  The  beneficial  effects  of 
salt  may  therefore,  and  in  the  long  run,  reside  not  so  much  in  the 
actual  sodium  and  chlorine  administered  as  in  the  additional  carbo- 
hydrate; fat,  or  protein  which  is  thus  rendered  available  for  the  main- 
tenance and  upbuilding  of  the  body. 

It  is  probably  for  some  such  reason  as  this  that  the  total  mineral- 
requirements  of  the  body  vary  exceedingly  with  the  dietary  upon  which 
an  animal  is  subsisting.'  Especially  is  this  the  case  when  the  require- 
ment on  a  normal  mixed  diet  is  contrasted  with  that  which  obtains 
when  the  diet  is  limited  in  such  a  way  as  to  provide  only  those  proteins 


40  INORGANIC  FOODSTUFFS 

of  vegetable  origin  which  are  most  remote  in  their  composition  from  the 
proteins  of  animal  tissue.  In  such  a  diet  a  large  proportion  of  the  nitro- 
gen is  wasted  because,  as  we  shall  see  in  a  subsequent  chapter,  the 
amino-acids  into  which  the  protein  splits  up  on  digestion  are  present 
in  the  wrong  proportion  and  have  to  be  resorted  and  selected  in  very 
different  proportions  in  order  to  build  up  proteins  of  the  animal  type. 
It  has  been  found  that  an  animal  subsisting  on  a  diet  of  this  kind  suffers 
not  only  a  large  wastage  of  nitrogen,  necessitating  the  consumption 
of  a  large  quantity  of  food  to  maintain  nitrogenous  equilibrium,  but 
also  a  large  wastage  of  mineral  constituents,  so  that  it  cannot  be 
maintained  in  health  or  nutritive  equilibrium  without  the  addition 
to  the  diet  of  a  considerable  excess  of  mineral  substances  over  the 
amount  which  would  be  required  by  an  animal  subsisting  on  a  more 
varied  diet. 

CALCIUM. 

During  the  early  months  of  the  growth  of  a  suckling  infant  or  animal, 
lime  is  very  rapidly  being  absorbed  and  utilized  by  the  tissues  for  the 
formation  of  bones.  This  calcium  is  totally  derived  from  milk.  Now 
the  lime  in  milk  is  present  therein  in  two  forms,  namely,  in  the  form 
of  calcium  phosphate  and  in  the  form  of  a  bulky,  indiffusible  compound 
with  one  of  the  proteins  of  milk,  casein.  The  calcium  phosphate  is,  of 
course  ionized,  but  the  calcium  caseinate,  on  the  contrary,  does  not 
yield  calcium  ions  in  solution. 

When  we  add  acids  to  milk,  or  when  owing  to  the  action  of  bacteria 
upon  the  milk-sugar  which  it  contains  lactic  acid  is  produced  in  the 
milk,  it  assumes  the  curdled  appearance  which  we  are  accustomed 
to  associate  with  "sour  milk."  This  appearance  is  due  to  the  separa- 
tion of  free  Casein,  uncombined  with  calcium,  which  has  been  abstracted 
from  the  calcium  caseinate  by  the  acid.  Free  casein  is  insoluble 
in  water  or  very  dilute  acids  and  hence  is  precipitated  in  curds  or 
flocculi,  while  the  calcium  is  now  present  in  the  "sour"  milk  in  the  form 
of  the  calcuim  salt  of  the  acid  which  has  been  added. 

Precisely  the  same  thing  happens  when  milk  which  has  been  ingested 
by  the  suckling  comes  into  contact  with  the  hydrochloric  acid  which 
is  contained  in  the  gastric  juice.  Free  casein;  more  or  less  modified  by 
partial  digestion,  is  precipitated  and  calcium  is  set  free  as  calcium 
chloride. 

There  has  been  a  good  deal  of  discussion  in  the  past  as  to  whether  the 
two  forms  of  lime  in  milk  are  equally  readily  utilized  by  the  suckling. 
In  view  of  the  above-mentioned  facts  there  would  appear  to  be  no  very 
good  reason  for  distinguishing  between  them,  since  in  the  stomach, 
where  absorption  begins,  both  forms  of  calcium  are  reduced  to  a 
common  level  by  the  conversion  of  the  calcium  caseinate  into  the 
ionized  and  diffusible  chloride. 

Notwithstanding  this  fact  it  has  been  frequently  argued  that  the 


CALCIUM  41 

calcium  which  is  combined  with  casein  in  milk  is  of  superior  nutritive 
value  to  that  which  is  present  in  the  milk  from  the  beginning  in  the 
form  of  diffusible  inorganic  salts  of  lime.  An  experiment  which  used 
to  be  frequently  quoted  in  support  of  this  view  was  that  of  Lunin's; 
who  fed  six  mice  upon  a  mixture  of  casem,  fat  and  cane-sugar  plus  the 
inorganic  salts  contained  in  milk.  These  animals  lived  respectively 
twenty,  twenty-three,  twenty-nine,  thirty  and  thirty-one  days;  where- 
as two  mice  of  the  same  age  fed  entirely  upon  whole  cow's  milk  for  a 
period  of  seventy-five  days  remained  in  good  health  at  the  end  of  the 
experiment.  In  the  first  experiment  the  inorganic  bases  were  all  com- 
bined with  inorganic  acids  to  form  diffusible  and  ionizable  salts,  where- 
as in  the  second  experiment  the  lime,  at  least,  was  combined  with 
casein.  Hence,  it  was  argued,  lime  in  the  inorganic  form  did  not  fulfil 
the  necessary  requirements  of  the  animals. 

This  experiment  might  easily  have  been  seen  from  the  first  to  be 
inconclusive,  for  natural  milk  and  an  artificial  mixture  such  as  that 
prepared  by  Lunin  must  obviously  differ  in  many  particulars  besides 
the  single  particular  of  the  diffusibility  of  the  calcium.  But  in  the 
light  of  our  more  recent  accessions  of  knowledge  concerning  the  nutri- 
tion of  animals  it  has  become  quite  clear  that  Lunin's  experiment 
bears  a  very  different  interpretation  to  that  which  was  originally  put 
upon  it. 

We  know  now,  thanks  to  researches  which  will  be  detailed  in  a  later 
part  of  the  work,  that  besides  a  sufficiency  of  proteins,  fat  and  carbo- 
hydrates, any  diet  which  is  to  maintain  animals  in  health  for  a  con- 
siderable period  must  contain  other  essential  constituents  which  are 
present  in  milk  or  in  animal  tissues  in  minute  amounts.  These  con- 
stituents fall  into  two  distinct  classes,  at  least,  and  possibly  as  our 
knowledge  increases  will  be  found  to  be  more  numerous  and  more 
diverse  in  their  chemical  characteristics  than  we  at  present  realize. 
The  two  classes  of  these  "accessory  foodstuffs'*  which  are  at  present 
recognized,  however,  are  in  the  first  place  the  vitamines,  which  are 
nitrogenous,  water-soluble  substances  and  in  the  second  place  a  group 
of  substances  which  are  commonly  found  associated  with  animal  fats, 
but  are  generally  absent  from  vegetable  fats.  Thus  Hopkins  has 
found  that  if  animals  be  fed  for  a  considerable  period  on  milk-salts, 
casein  and  milk-sugar  they  will  not  survive,  while  the  addition  of  a 
small  amount  of  butter  suffices  to  render  the  diet  adequate  for  the 
needs  of  the  animals. 

In  the  light  of  these  facts  it  will  readily  be  seen  that  Lunin's  experi- 
ment does  riot  bear  on  the  question  of  calcium-nutrition  at  all,  but 
rather  on  the  question  of  accessory  .organic  foodstuffs.  Furthermore, 
recent  experiments  have  shown  that  the  cane-sugar  employed  by  Lunin 
in  his  artificial  mixture  is  not  by  any  means  a  sufficient  substitute 
for  milk-sugar  in  the  dietary  of  young  animals. 

There  is  thus  no  evidence  whatever  that  the  two  forms  of  lime  in 
milk  are  not  equally  available  and  useful  to  the  suckling,  as  we  should 


42  INORGANIC  FOODSTUFFS 

expect  them  to  be  from  the  fact  that  they  are  alike  diffusible  and 
ionizable  very  shortly  after  they  arrive  within  the  stomach. 

These  considerations  have  an  important  bearing  upon  the  practical 
Question  of  the  modification  of  cow's  milk  for  infant-feeding.  It  is 
me  common  practice  to  add  lime-water  (calcium  hydroxide  solution) 
to  milk  for  young  infants  for  two  purposes;  in  the  first  place  in  order 
to  delay  the  acidification  and  consequent  "curdling"  of  the  milk  by  the 
hydrochloric  acid  in  the  stomach.  This  results  in  deferring  the  floc- 
culation  of  the  casein  until  it  has  undergone  partial  digestion  by  the 
rennin  and  pepsin  in  the  gastric  juice,  when  the  flocculi  which  are 
formed  are  finer  and  more  gelatinous  and  therefore  more  easily  pene- 
trable by  digestive  juices  than  they  are  if  curdling  occurs  without  pre- 
liminary digestion.  In  the  second  place  the  lime-water  is  added  with  a 
view  to  increasing  the  supply  of  lime  to  the  infant  and  thus  assisting 
the  growth  of  bony  tissues,  teeth,  etc.  From  the  latter  point  of  view 
this  practice  has  been  decried  in  some  quarters,  on  the  ground  that 
lime  which  is  not  organically  combined  is  not  so  readily  assimilated 
and  utilized  as  calcium  which  is  in  organic  combination.  We  have 
seen  that  there  is  no  experimental  justification  for  this  distinction,  and 
even  if  there  were,  the  lime-water  which  is  added  to  milk  immediately 
combines  in  considerable  proportion  with  the  casein  to  form  a  com- 
pound of  exactly  the  same  type,  only  richer  in  calcium,  as  that  which  is 
found  in  normal  milk,  so  that  the  greater  part  of  the  calcium  thus 
administered  does  in  fact  reach  the  stomach  in  a  state  of  organic 
combination. 

It  is,  of  course,  quite  another  question  whether  administration  of 
lime  beyond  a  certain  daily  amount  is  of  any  value  in  assisting  the 
growth  of  bony  tissues.  Experience  in  connection  with  other  articles 
of  diet  conclusively  shows  us  that  in  many  instances  the  effective 
administration  of  foodstuffs  is  limited  by  the  ability  of  the  tissues  to 
utilize  and  elaborate  them,  any  supply  in  excess  of  this  being  rejected 
and  wasted.  Defective  development  of  bony  tissues  may  be  sometimes 
attributable  to  deficiency  of  lime  in  the  diet,  but  it  is  probably  more 
often  due  to  inability  of  the  bone-producing  tissues  to  utilize  the  lime 
which  is  presented  to  them.  This,  however,  obviously  constitutes  no 
objection  to  the  addition  of  lime-water  to  the  milk  of  an  infant;  it 
merely  indicates  a  reason  why  this  procedure  by  itself  may  often  be 
insufficient  to  correct. faulty  or  deferred  development  of  the  calcareous 
tissues. 

The  lime-requirement  of  the  adult  is  very  greatly  increased  in  the 
female  by  activity  of  the  mammary  glands.  Thus  from  0.3  to  0.5 
gramme  of  calcium  oxide  per  hundred  pounds  of  body-weight  per  day 
is  sufficient  to  supply  the  minimum  needs  of  a  pig  or  goat  which  is  not 
yielding  milk,  but  a  milch-goat  requires  an  additional  1  to  2  grammes 
of  calcium  oxide  per  day  for  every  pound  of  milk  it  yields.  Insufficiency 
of  lime  in  the  diet  under  such  circumstances  results  in  actual  withdrawal 
of  lime  from  the  skeleton,  a  condition  which  when  it  becomes  sufficiently 


IRON  43 

acute  to  cause  softening  and  bending  of  the  bones  is  known  as  Osteo- 
malacia.  It  is  not  to  be  inferred,  however,  that  osteomalacia  is  always 
due  to  deficiency  of  calcium  in  the  diet.  It  may  be  due  as  indicated 
above  to  physiological  disturbances  or  nutritional  deficiencies  leading- 
to  faulty  utilization  of  the  calcium  which  the  dietary  affords. 

Calcium  is  excreted,  in  part  by  the  kidneys  and  in  part  by  the 
intestinal  mucosa.  A  high  proportion  of  soluble  phosphates  in  the 
diet  tends  to  increase  the  output  of  calcium  in  the  feces,  probably  owing 
to  the  formation  of  calcium  phosphate  which  is  insoluble  in  the  alkaline 
fluids  of  the  intestine.  Just  as  potassium  salts  increase  the  output  of 
sodium  in  the  urine,  so,  and  for  similar  reasons,  do  magnesium  salts 
increase  the  output  of  calcium  in  the  urine. 

IRON. 

Iron  is  an  essential  constituent  of  the  red  pigment  of  the  blood, 
Hemoglobin.  Since  hemoglobin  is  the  carrier  of  oxygen  from  the 
lungs  to  the  tissue-cells,  it  is  obvious  that  iron  in  this,  if  in  no  other 
capacity,  plays  a  vital  part  in  the  economy  of  the  body,  but,  in  addition 
to  the  hemoglobin-iron,  iron  is  also  found,  and  not  necessarily  asso- 
ciated with  hemoglobin,  in  other  parts  of  the  body.  Thus  the  liver 
contains  about  0.02  per  cent,  of  iron  calculated  on  the  basis  of  the 
fresh,  undried  organ  washed  free  from  blood.  The  muscles  contain 
appreciable  quantities  of  iron,  especially  heart-muscle,  which  contains 
about  0.01  per  cent,  of  the  fresh,  undried  weight.  In  smaller  quantities 
iron  is  found  elsewhere  in  the  body,  regularly  accompanying  Nucleins 
and  Nucleoproteins  wherever  they  are  found. 

The  iron-content  of  the  adult  is  subject,  like  that  of  other  tissue- 
constituents,  to  daily  losses.  Experiments  with  starving  individuals 
(and  it  is  under  conditions  of  starvation  that  the  body  is  most  economi- 
cal of  its  resources)  show  that  the  nominal  daily  loss  of  iron  in  the 
feces  is  from  seven  to  eight  milligrammes,  while  in  addition  to  this  a 
daily  loss  of  about  one  milligramme  occurs  through  the  kidneys.  In 
all,  then,  it  is  probable  that  about  ten  milligrammes  of  iron,  or  about 
one  three-hundredth  of  the  total  hemoglobin-iron  in  the  body  is  lost 
per  day.  This  loss  must  be  replaced  from  the  diet. 

Under  certain  pathological  conditions,  or  conditions  of  malnutrition, 
a  loss  of  hemoglobin  occurs  from  the  blood  and  the  patient  is  said  to 
have  become  "anemic."  This  loss  of  hemoglobin  may  and  on  the 
other  hand  may  not  be  accompanied  by  a  diminution  in  the  number  of 
red  blood-corpuscles.  As  might  be  anticipated,  the  result  of  this 
condition  is  suboxidation  in  the  tissues  with  consequent  symptoms 
which  are  sometimes  of  the  severest  gravity.  These  are  very  well 
illustrated  by  the  chlorosis,  or  "green  sickness"  which  very  frequently 
overtakes  girls  at  the  age  of  puberty.  From  periods  of  remote  antiquity 
antedating  by  many  centuries  our  knowledge  of  the  chemical  compo- 
sition and  significance  of  hemoglobin,  this  disease  has  been  combated 


44  INORGANIC  FOODSTUFFS 

by  the  administration  of  inorganic  salts  of  iron,  and  often  with  beneficial 
effect.  For  long  it  was  thought,  without  any  question,  that  the  salts 
of  iron  so  administered  were  absorbed  and  that  the  beneficial  effect  of 
the  medicament  was  due  to  the  replacement  of  the  iron  in  the  blood  by 
the  iron  so  administered.  Doubt  was  thrown  upon  this  explanation 
by  the  discovery  that  iron  is  eliminated  from  the  body  in  the  feces. 
Doses  of  inorganic  salts  of  iron,  administered  to  healthy  individuals, 
were  recovered  apparently  unaltered  in  the  feces,  and  from  this  fact 
the  erroneous  conclusion  was  drawn  that  inorganic  salts  of  iron  are  not 
absorbed.  The  beneficial  effects  of  iron  in  anemia  were  either  denied, 
a  denial  in  which  practising  physicians  declined  to  share,  or  else 
accounted  for  by  the  irritant  action  of  the  salts  of  iron  upon  the  epithe- 
lium of  the  intestinal  tract.  A  mild  irritation  has  a  well-known  "  tonic" 
effect  which  is  rather  difficult  to  define  in  precise  terms;  but  which  is 
frequently  manifested,  not  only  by  increased  activity  of  the  tissues 
which  are  stimulated,  but  also  of  other  and  sometimes  distant  tissue. 
The  beneficial  effects  of  iron  were  therefore  attributed  to  increased 
activity  of  the  tissues  resulting  in  increased  assimilation  and  utilization 
of  the  organically  combined  iron  in  the  diet  and  not  to  direct  assimi- 
lation of  the  iron  administered  as  a  medicament. 

Much  has  been  done  to  clear  up  this  question  by  the  employment  of 
microchemical  tests  to  trace  the  course  of  iron  through  the  intestine. 
When  mice  are  fed  upon  milk  alone  for  a  considerable  period,  on  placing 
the  alimentary  canal  of  these  animals  in  ammonia  and  ammonium  sul- 
phide the  characteristic  precipitate  of  iron  sulphide  does  not  appear, 
or  at  the  most  there  is  only  a  very  slight  green  coloration.  Now  milk 
is  one  of  the  articles  of  diet  which  is  poorest  in  iron,  cow's  milk  con- 
taining only  about  2.3  mg.  of  iron  per  100  grammes  of  dry  substance. 
Very  different  results  are  obtained  if  the  mice  are  fed  upon  milk  to  which 
inorganic  salts  of  iron  have  been  added.  In  the  stomach  there  is  little 
if  any  reaction  for  iron,  while  in  the  duodenum  there  is  a  marked  green 
coloration.  If  the  tissues  of  the  intestine  are  examined  under  the  micro- 
scope, little  granules  of  iron  are  found  imbedded  in  the  protoplasm 
of  the  intestinal  epithelium,  and  leukocytes  are  found  laden  with 
minute  particles  of  iron.  In  the  jejunum,  however,  and  in  the  ileum, 
very  little  iron  is  found,  while  in  the  cecum  and  large  intestine  a  strong 
iron-test  is  once  more  obtained. 

Coming  from  the  intestinal  canal,  especially  from  the  duodenum, 
the  lymphatics  may  be  seen  filled  with  cells  containing  iron.  The  liver 
and  spleen  give  much  stronger  tests  for  iron  than  those  of  the  mice 
fed  upon  milk  alone. 

There  can  be  no  question,  therefore,  but  that  the  inorganic  iron- 
salts  thus  administered  are  absorbed.  Part  of  the  iron  appears  to  be 
conducted  by  way  of  the  lymphatics  to  the  thoracic  duct  and  the  blood- 
stream. Part  is  unquestionably  conducted  by  the  portal  vein  to  the 
liver,  which  is  a  storehouse  of  iron  as  it  is  of  many  other  things .  The 
absorption  takes  place  mainly  in  the  duodenum;  the  excretion  of  waste 


IRON  45 

iron  occurring,  on  the  contrary,  in  the  cecum  and  large  intestine, 
although  part  of  the  small  intestine  may  also  participate  in  this 
function. 

It  is  one  thing  to  show  that  inorganic  salts  of  iron  are  absorbed  and' 
it  is  another  to  show  that  they  may  be  utilized  in  the  building  up  of 
hemoglobin.  The  iron  in  hemoglobin  is  very  firmly  and  intimately 
combined,  and  cannot  be  detected  by  the  reagents  ordinarily  employed 
for  this  purpose,  such  as  ammonium  sulphide  or  potassium  ferro- 
cyanide.  In  fact  the  iron  in  hemoglobin  resists  the  action  of  boiling, 
concentrated  potassium  hydroxide  and  boiling  hydrochloric  acid.  Only 
by  dissolving  the  hematin  radical  (which  is  the  iron-containing  moiety 
of  the  hemoglobin  molecule)  in  concentrated  sulphuric  acid  is  the 
iron  split  off  and  the  hematin  changed  into  iron-free  hematin,  or 
Hematoporphyrin. 

Most  of  the  iron  in  our  diet  is  in  the  form  of  hemoglobin  or  other 
organic  compounds  of  iron  from  which  free  ionic  iron  is  not  readily 
split  off.  The  yolk  of  eggs  is  very  rich  in  iron,  as  might  be  anticipated 
from  the  fact  that  the  yolk  of  an  egg  must  contain  all  of  the  constit- 
uents necessary  to  form  the  hemoglobin  of  the  developing  embryo. 
The  iron-compound  in  yolks  of  eggs  is  not  hemoglobin,  but  some 
antecedent  of  hemoglobin.  On  extracting  the  yolk  of  a  hen's  egg  with 
alcohol  or  ether,  none  of  the  iron  goes  into  the  extract.  The  residue, 
which  contains  all  of  the  iron,  is  a  mixture  of  proteins  and  nucleo- 
proteins.  The  iron  cannot  be  extracted  from  this  residue  by  alcohol 
and  hydrochloric  acid,  although  inorganic  salts  of  iron  readily  yield 
up  iron  to  these  reagents.  During  the  digestion  of  iron-containing 
protein  by  Pepsin  in  the  stomach,  the  part  containing  iron  does  not  go 
into  solution  and  its  digestion  is  not  accomplished  until  it  reaches  the 
small  intestine  and  comes  in  contact  with  the  digestive  fluid  secreted 
by  the  pancreas.  It  is  not  digestible  by  pepsin  and  in  this  and  in  other 
respects  corresponds  in  its  behavior  to  the  class  of  bodies  which  the 
reader  will  later  learn  to  recognize  as  nucleins.  The  ordinary  tests  for 
iron  are  given  by  this  substance,  to  which  von  Bunge  gave  the  name 
"Hematogen,"  but  not  so  readily  as  by  inorganic  salts  of  iron.  On 
adding  ammonium  sulphide  to  an  ammoniacal  solution  of  this  nuclein 
a  greenish  coloration  is  produced,  which  only  slowly  changes  to  black 
on  standing.  In  other  words  ionized  iron  is  at  first  only  present  in 
traces  and  is  slowly  split  off  from  the  compound  under  the  prolonged 
influence  of  the  reagents.  The  compound  thus  behaves  in  a  manner 
very  like  that  of  the  protein  salts  of  the  heavy  metals,  for  instance 
casein  salts  of  silver,  mercury  and  so  forth  to  which  the  reader's  atten- 
tion will  be  directed  in  a  later  chapter.  There  is  little  reason  to  doubt 
that  hematogen  is  simply  a  protein  salt  of  iron  in  which  the  protein  is 
acting  the  part  of  a  weak  acid,  or  else  a  double  salt  of  protein  and  an 
inorganic  salt  of  iron.  Protein  compounds  of  this  type  yield  no  metal- 
ions  in  solution,  or  at  the  most,  only  traces  of  them. 

Since  compounds  such  as  these  are  the  only^forms  in  which  we 


46  INORGANIC  FOODSTUFFS 

normally  receive  iron  in  our  diet,  for  we  only  partake  of  inorganic 
salts  of  iron  as  a  therapeutic  measure,  there  can  be  no  question  but  that 
we  can  absorb,  assimilate  and  utilize  the  iron  contained  in  organic, 
non-ionized  compounds. 

It  will  be  recollected  that  the  iron  in  hemoglobin  or  hematin  does 
not  yield  the  ammonium-sulphide  test  for  iron.  On  administering 
hematin  or  hemoglobin  to  mice  which  have  undergone  iron-starvation, 
however,  and  applying  the  iron-sulphide  test  to  various  parts  of  the 
intestine,  we  ascertain  the  remarkable  fact  that  the  duodenum  and  the 
cecum  yield  the  iron-test  just  as  they  do  when  inorganic  iron  is  admin- 
istered. In  other  words,  the  iron  in  the  process  of  digestion  in  the 
duodenum  has  become  loosened  from  its  combination  in  the  hematin 
radical  and  set  free  as  an  inorganic  or  at  least  an  ionized  salt  of  iron. 
Since  the  iron,  immediately  subsequent  to  absorption,  appears  in  the 
same  condition  whether  administered  in  the  ionic  form  or  not,  there 
would  appear  to  be  no  very  good  reason  for  supposing  that  inorganic 
salts  of  iron  are  not  utilized  to  nearly  as  great  an  extent  as  the  organic 
salts  of  iron.  The  most  specific  disadvantage  which  attends  the  use 
of  inorganic  salts  of  iron  is  their  irritating  or  corrosive  action  upon  the 
intestinal  epithelium,  a  corrosive  action  which,  like  that  of  mercury 
salts,  is  probably  to  be  attributed  to  the  formation  of  insoluble  protein 
salts  of  the  metal  within  the  epithelial  cells.  This  leads  to  the  dis- 
ruption of  the  gelatinous  structures  of  the  cells  and  their  conversion 
into  granules  or  flocculi  which,  no  longer  being  held  together  by  the 
cohesiveness  of  a  jelly,  fall  apart  with  consequent  disintegration  of  the 
cells.  Many  individuals  who  display  an  "  idiosyncrasy"  or  exceptional 
sensitiveness  to  intestinal  irritation  are  very  severely  affected  by  this 
corrosive  action  of  iron-salts  and  for  this  reason  the  general  employment 
of  non-ionized  organic  compounds  of  iron  in  therapeutics,  such  as 
hemoglobin  or  hematogen,  is  much  to  be  preferred. 

With  the  exception  of  the  disadvantages  arising  from  the  corrosive 
action  of  inorganic  salts  of  iron,  therefore,  the  ionized  and  unionized 
compounds  would  appear,  so  far  as  the  above-cited  evidence  goes,  to 
be  equally  useful  sources  of  iron  in  the  diet.  There  are  certain  impor- 
tant facts,  however,  which  would  appear  at  first  sight  to  bear  out  the 
contention  that  inorganic  salts  of  iron,  notwithstanding  their  absorp- 
tion, are  not  utilizable  for  the  synthesis  of  hemoglobin.  We  have 
seen  that  milk  contains  a  very  low  percentage  of  iron  in  comparison 
with  other  foods,  especially  in  comparison  with  green  vegetables, 
certain  fruits  such  as  apples,  and  flesh.  If  sucklings  are  kept  beyond 
the  normal  period  of  lactation  exclusively  upon  a  milk  diet,  they  become 
anemic  from  lack  of  iron.  If  we  compare  rabbits  which  have  been 
allowed  to  change  to  a  diet  of  green  vegetables  after  the  normal  period 
of  lactation,  with  those  which  have  been  brought  up  upon  an  exclusive 
milk-diet,  we  find  that  the  former  contain  much  more  hemoglobin  than 
the  latter.  But  the  remarkable  fact  is  that  if  we  add  inorganic  salts 
of  iron  to  the  milk-diet  the  total  hemoglobin  in  the  animals  is  not 


IRON  47 

increased,  although  they  grow  much  more  rapidly  than  the  similarly 
fed  animals  which  do  not  receive  iron.  This  would  appear  to  indicate 
that  inorganic  salts  of  iron  are  utilizable  for  certain  purposes  in  the 
body  connected  with  the  growth  of  the  animals,  but  not  for  the  building 
up  of  hemoglobin.  This  conclusion,  however,  would  be  premature. 

Recent  acquisitions  to  our  knowledge  of  the  structure  of  the  hematin 
moiety  of  the  hemoglobin  molecule  have  shown  that  it  contains  a 
particular  molecular  grouping,  namely,  the  Pyrrole  Group : 

__c  —  c— 

II      II 
— c  — c— 

\/ 

N 

which  there  is  every  reason  for  supposing  cannot  be  synthesized  by 
animals  but  must  be  obtained  by  them  preformed,  that  is  to  say  from 
the  tissues  of  plants  or  from  the  tissues  of  animals  which  acquired  it 
from  plants.  This  pyrrole  grouping  is  contained  in  small  amounts  in 
the  majority  of  proteins  and  it  forms  a  very  important  component  of 
Chlorophyll,  the  green  coloring-matter  of  plants  which,  as  we  shall  see, 
is  very  closely  related,  chemically,  to  hemoglobin.  It  is  not  improb- 
able, therefore,  that  inorganic  iron-salts  added  to  an  exclusive  milk- 
diet  are  not  utilized  for  building  up  hemoglobin  simply  for  the  reason 
that  other  component  parts  of  the  hemoglobin  molecule,  as  essential  as 
iron  itself,  are  either  lacking  altogether  in  the  milk-diet  or  present 
therein  in  insufficient  amount  to  subserve  the  needs  of  the  blood-form- 
ing tissues  and  those  of  the  other  tissues  of  the  body  as  well.  We  will 
return  to  this  question  in  later  chapters  in  connection  with  the 
chemistry  of  hemoglobin,  and  again  in  connection  with  the  general 
problems  of  growth  and  nutrition. 

The  percentages  of  iron  which  are  contained  in  several  common 
articles  of  food  are  enumerated  in  the  following  table: 

IRON-CONTENT  OF  FOODS  IN  PER  CENT.  OF  EDIBLE  PORTION,  AFTER 

SHERMAN.1 


Food. 
Egg-white    . 

Iron  (Fe). 
0  0001 

Food. 
Potatoes 

Iron  (Fe). 
0  0013 

Butter    .... 
Whole  milk       .      . 

.      .      .      0.0002 
0  00024 

Cheese    . 
Dates 

0.0013 
0  0030 

Apples   .... 
Carrots 
Lettuce 
Cornmeal    . 
White  bread      . 
Asparagus 
Cabbage 
Fish 

.      .      .      0.0003 
.      .      .      0.0006 
.      .      .      0.0007 
.      .      .      0.0009 
.      .      .      0.0009 
.      .      .      0.0010 
.      .      .      0.0011 
0.0008-0.0013 

Eggs        .,   -. 
Meat 
Spinach  . 
Oatmeal 
Barley    .      . 
Egg-yolk 
Blood      .      . 

......      0.0030 
.      .      .      .   0.0023-0.0033 
0.0036 
.      ....      0.0038 
0.0041 
0.0086 
0.0526 

It  will  be  noted  that  the  iron-content  of  spinach  is  very  high.  Spin- 
ach is  also  very  rich  in  chlorophyll,  as  its  deep  green  color  indicates, 
and  thus  contains  a  large  proportion  of  another  essential  constituent 

1  Chemistry  of  Food  and  Nutrition,  New  York,  1918. 


48  INORGANIC  FOODSTUFFS 

of  hemoglobin,  the  pyrrole  radical.  Chlorophyll,  it  is  true,  is  indigest- 
ible by  the  digestive  juices,  but  it  is  split  up  by  the  bacteria  which 
inhabit  the  intestine,  and  in  this  way  a  portion  of  the  pyrrole  which  it 
contains  may  possibly  be  rendered  available  for  assimilation  from  the 
intestine  and  utilization  by  the  tissues. 

It  will  be  recollected  that  if  iron  is  administered  to  young  animals 
which  are  undergoing  iron-starvation  by  being  kept  upon  an  exclusive 
milk-diet,  their  growth  is  markedly  accelerated  despite  the  fact  that 
the  iron  is  not  utilized  to  build  up  hemoglobin.  This  effect  is  of  sig- 
nificance, inasmuch  as  it  indicates  that  iron  subserves  other  important 
functions  in  the  body  besides  that  of  entering  into  the  composition 
of  the  oxygen-carrying  pigment  of  the  blood.  We  are  reminded  of  the 
prevalence  of  iron  in  nuclear  elements,  and  led  to  suspect  that  iron 
plays  some  essential  part  in  the  functions  of  the  nuclei.  It  is  a  note- 
worthy fact,  however,  that  if  iron  be  added,  in  similar  amounts  to  those 
employedjn  the  above-cited  experiments,  to  an  abundant  and  mixed 
diet,  containing  a  normal  sufficiency  of  iron,  this  acceleration  of  growth 
is  not  observed.  Evidently  beyond  a  certain  diurnal  allowance  the 
tissues  of  the  growing  animal  are  not  able  to  utilize  iron  for  the  purposes 
which  result  in  the  acceleration  of  growth.  Here  we  meet  again  with  a 
phenomenon  to  which  reference  was  made  in  connection  with  the  utili- 
zation of  calcium.  The  ability  of  the  tissues  to  profitably  utilize  the 
materials  brought  to  them  sets  a  definite  limit  to  the  amount  of  a  food- 
stuff which  it  is  of  any  avail  to  consume.  It  is  doubtless  for  this 
reason  that  iron,  whether  in  the  organic  or  the  inorganic  form,  is  with- 
out effect  in  accelerating  the  rebuilding  of  hemoglobin  after  hemor- 
rhage. The  blood-forming  tissues  are  able  to  manufacture  so  much 
hemoglobin  per  diem  and  the  supply  of  more  raw  materials  than  they 
can  "work  up"  in  a  day  is  useless. 

The  ultimate  reason  for  this  phenomenon,  which  is  of  such  general 
occurrence  in  life-phenomena,  resides  undoubtedly  in  the  multifarious 
variety  of  the  chemical  processes  which  underlie  and  accompany  vital 
activities.  In  every  detail  of  change  which  accompanies  the  per- 
formance of  any  function  by  living  tissues  not  merely  one  chemical 
reaction  is  involved  but  a  whole  series  of  interwoven  reactions  following 
and  depending  upon  one  another.  Now  in  any  series  of  chemical 
changes  of  which  the  second  utilizes  some  product  of  the  first,  the  third 
some  product  of  the  second,  and  so  forth,  it  is  always  the  specifically 
slowest  reaction  which  "sets  the  pace"  for  those  which  succeed  it. 
No  matter  how  quickly  raw  materials  may  be  supplied,  this  "master- 
reaction"  can  proceed  only  at  a  certain  maximum  speed  and  succeeding 
reactions  must  wait  for  its  products  before  they  can  seize  and  elaborate 
them.  Provided  then,  that  any  article  of  diet  be  supplied  in  sufficiency 
to  maintain  at  top  speed  the  "master-reaction"  of  the  series  of  pro- 
cesses intojwhich  it  enters,  excess  of  this  particular  item  in  the  dietary 
is  mere  wastagejand  casts  an  unnecessary  strain  upon  the  organs  of 
elimination. 


OTHER  INORGANIC  FOODSTUFFS  49 

Insufficient  hemoglobin  content  of  the  blood,  therefore,  and  any 
other  type  of  maldevelopment  and  malnutrition  may  originate  in 
either  of  two  ways,  namely,  through  inadequacy  of  the  diet,  or  through 
imperfect  utilization  of  substances  which  are  present  in  abundance  in 
the  dietary.  Certain  mild  types  of  anemia,  probably  belong  to  the 
former  category  and  the  consensus  of  opinion  of  the  physicians  is 
that  these  are  favorably  affected  by  administration  of  iron.  In  other 
types  of  anemia,  in  which  the  utilization  of  iron  is  defective  or  in  which, 
as  in  the  anemia  of  hemorrhage,  the  lack  of  hemoglobin  is  due  to 
loss  or  destruction  after  it  has  been  manufactured,  we  cannot  expect 
therapeutic  administration  of  iron  to  be  followed  by  equally  favorable 
results. 

OTHER  INORGANIC   FOODSTUFFS. 

The  remaining  inorganic  constituents  of  the  body  will  be  but  briefly 
considered  at  this  point,  some  of  them  falling  under  review  in  other 
connections  in  later  chapters.  While  tjie  majority  of  them  probably 
play  important  or  even  essential  parts  in  our  bodily  economy,  we  have 
as  yet  only  succeeded  in  a  few  instances  in  obtaining  a  clue  to  the 
nature  of  these  functions. 

Among  the  metals  other  than  those  which  we  have  considered, 
Magnesium  is,  from  a  quantitative  point  of  view,  the  most  important. 
Magnesium  is  found  in  small  quantities  in  all  animal  and  plant 
cells,  and  in  milk.  There  appears  to  be  a  rather  definite  relation- 
ship or  proportionality  between  the  magnesium  and  the  calcium 
contents  of  the  tissues,  and  from  the  fact  that  a  trifling  excess  of 
magnesium,  when  introduced  into  the  circulation,  causes  profound 
disturbances  such  as  glycosuria,  we  may  conclude  that  magnesium  has 
powerful  physiological  actions  and  that  in  consequence  even  the 
amounts  which  normally  occur  in  tissues  are  not  devoid  of  physiological 
significance. 

It  is  stated  that  traces  of  Lithium  are  normally  found  in  animal 
tissues,  and  it  is  a  much-discussed  question  whether  or  not  a  minute 
trace  of  Arsenic  is  a  normal  constituent  of  human  tissues,  the  gravity 
of  the  discussion  being  attributable,  of  course,  to  the  medicolegal 
significance  of  the  question.  The  consensus  of  opinion  appears 
to  be,  however,  that  arsenic  is  found  in  human  tissues  only  after  the 
administration  of  drugs  containing  arsenic  or  in  districts  where  arsenic 
occurs  in  considerable  amounts  in  the  soil  and  water. 

Among  non-metallic  inorganic  constituents  of  the  body,  Chlorine 
plays  a  leading  part,  in  the  alkali  chlorides  of  the  blood  and  tissues 
and  in  the  hydrochloric  acid  in  the  gastric  juice.  It  is  derived  from 
chlorides  in  the  food. 

Fluorine  occurs  in  small  amounts  in  milk  (0.00003  per  cent.)  and 
is  a  normal  constituent  of  bones  and  teeth;  it  is  unquestionably  not 
devoid  of  significance  in  the  formation  of  these  tissues. 
4 


50  INORGANIC  FOODSTUFFS 

Silicon  is  a  constant  constituent  of  hair  and  feathers,  no  less  than 
40  per  cent,  of  the  ash  of  hair  consisting  of  SiO2.  This  is  doubt- 
less derived  from  silicates  in  the  vegetable  portion  of  our  diet, 
silicon  playing  an  important  part  in  communicating  rigidity  to  many 
plant-tissues.  According  to  Drechsel  the  silicon  in  feathers  exists 
therein  in  a  state  of  organic  combination,  as  the  silicate  of  a  hydro- 
aromatic  alcohol  closely  related  to  cholesterol. 

Phosphorus  is,  of  course,  an  element  of  prime  importance  in  the 
life-economy,  in  the  form  of  the  phosphoric  acid  radical  in  phospho- 
proteins  such  as  casein  and  in  the  form  of  complex  substituted 
phosphoric  acids,  as  Nucleic  Acid  and  the  glycero-phosphoric  acid 
radical  of  the  phosphorus-containing  fats  or  phospholipins.  This 
phosphorus  is  derived  from  the  phosphates,  phosphoproteins,  nucleins 
and  phospholipins  in  the  diet.  There  is  some  room  for  question 
whether  animal  tissues  utilize  the  inorganic  phosphates  in  the  diet 
for  the  building  up  of  the  nucleins  and  phospholipins.  A  fact  which 
seems  to  indicate  that  animals  do  not  depend  upon  inorganic  phosphates 
for  the  production  of  these  substances  is  that  mice  will  grow  normally 
and  reproduce  on  a  diet  containing  a  high  proportion  of  aluminum 
hydrate,  although  this  results  in  the  formation  of  the  insoluble  alumi- 
num phosphate  from  any  inorganic  phosphates  which  may  be  present 
in  the  alimentary  canal,  and  its  elimination,  without  absorption  in  the 
feces. 

Sulphur  also  plays  an  exceedingly  important  role,  but  in  the  form  of 
the  complex  amino-acid  cystine,  which  is  a  decomposition-product 
of  many  proteins,  rather  than  in  the  form  of  free  sulphates  or  sulphides. 

Iodine  is  a  normal  constituent  of  the  Thyroid  and  plays  an  essential 
part  in  the  important  functions  of  this  gland.  We  will  consider 
the  nature  of  the  organic  combination  in  which  it  occurs  and  its 
significance  in  the  bodily  economy  in  a  later  chapter.  It  has  been 
repeatedly  stated  that  iodine  is  found  in  other  tissues  of  the  body, 
notably  in  the  pituitary  gland,  but  more  recent  analyses  have  shown 
that  in  the  absence  of  iodide-medication,  iodine  is  not  found  in  normal 
animal  tissues  other  than  the  thyroid.  Iodine  is  an  important  con- 
stituent of  seaweed,  from  the  ash  of  which  a  quantity  of  the  iodine  of 
commerce  is  derived.  The  relatively  high  concentration  of  iodine  in  the 
tissues  of  these  marine  plants  is  of  especial  interest  because  the  iodine 
content  of  sea-water  is  exceedingly  low.  This  constitutes  therefore  an 
interesting  case  of  the  Selective  Absorption  by  living  tissues  to  which 
reference  was  made  in  connection  with  the  proportion  of  sodium  to 
potassium  in  the  blood  and  tissues  of  animals. 

THE   COMPLEXITY   OF   OUR  DIETARY   REQUIREMENTS. 

It  is  to  be  hoped  that  the  recital  of  the  above  category  of  the  inorganic 
constituents  of  our  body,  present,  several  of  them,  in  the  most  incon- 
siderable traces,  will  have  the  effect  of  making  the  reader  pause  ere  he 


COMPLEXITY  OF  OUR  DIETARY  REQUIREMENTS         51 

embraces  any  of  the  dietary  fads  and  "systems"  which  are  so  prevalent 
in  this  uninformed  and  loquacious  period  of  our  social  evolution.  The 
average  man  or  woman  hesitates  to  pronounce  an  opinion  on  the  motive 
machinery  of  steamships  or  aeroplanes  or  on  the  fuel-requirements  of 
a  Diesel  engine,  but  regarding  that  infinitely  more  complex  engine,  a 
human  being,  the  average  individual  deems  himself  fully  informed  and 
all  that  is  required  to  make  numerous  converts  to  any  dietetic  fad  is  a 
considerable  degree  of  self-assurance. 

So  complex  are  the  requirements  of  the  animal  economy;  so  little 
do  we  know  the  parts  that  these  several  requirements  play  and  their 
delicate  adjustments  to  one  another,  that  we  are  totally  unable  at  this 
stage  of  our  knowledge  to  enumerate  the  constituents  of  any  restricted 
dietary  which  shall  certainly  and  for  prolonged  periods  of  time,  convey 
to  the  subject  all  that  he  requires  for  the  orderly  functioning  of  his 
body.  In  medical  practice  it  is,  of  course,  necessary  to  occasionally 
prescribe  a  limited  and  specified  diet  for  a  definite  period  in  order  to 
combat  certain  conditions  or  maladies,  but  to  do  so  for  lengthy  periods 
of  time,  especially  for  growing  infants  and  children,  is  to  simply  assume 
a  knowledge  which  we  do  not  possess.  The  problem  of  the  dietary 
requirements,  as  we  have  seen,  is  complex  enough  when  we  consider 
only  the  inorganic  foodstuffs;  but  when  we  add  to  these  the  organic 
requirements  of  the  body  the  complexity  of  the  problem  of  nutrition 
is  multiplied  a  hundredfold,  and  we  are  as  yet  hopelessly  in  the  dark 
respecting  the  source  and  function  of  a  multitude  of  constituents  of  the 
body  and  of  the  degree  to  which  they  may  be  essential.  Our  knowl- 
edge in  this  field  is  rapidly  extending,  perhaps  more  rapidly  at  present 
than  in  any  other  field  of  biochemistry,  but  even  at  the  present  rate 
of  accession  of  knowledge,  the  complete  knowledge  essential  for  enumer- 
ation in  detail  of  all  the  dietary  requisites  of  a  human  being  is  very 
far  distant  indeed. 

The  knowledge  that  we  do  possess,  however,  enables  us  in  certain 
particular  instances,  as,  for  example,  in  the  Weir  Mitchell  treatment 
of  certain  nervous  disorders,  or  the  Allen  treatment  of  diabetes,  to 
accomplish  very  decisive  therapeutic  results  by  restricted  dietaries 
prescribed  for  limited  periods,  in  conjunction  with  hygienic  measures 
and  adequate  biochemical  and  clinical  observation  and  control.  The 
very  success  of  such  measures  in  any  particular  instance  carries  with 
it  the  danger  of  converting  an  ignorant  patient  into  a  fanatical  diet- 
faddist  who,  upon  recovery  of  health,  proceeds  to  convert,  first  his 
acquaintances  and  then,  if  he  has  the  opportunity,  a  wider  public,  to  the 
health  doctrine  which  he  has  evolved  out  of  the  temporary  measures 
of  the  physician.  This  is  no  doubt  the  origin  of  many  of  the  dietary 
and  hygienic  eccentricities  to  which  certain  genuine  or  imaginary 
invalids  devote  themselves.  No  small  part  of  this  perverted  activity 
could  probably  be  stifled  at  its  birth,  if  the  physician  who  is  prescribing 
dietary  or  hygienic  measures  were  to  make  a  practise  of  explaining  as 
thoroughly  and  simply  as  he  is  able,  to  the  patient  and  his  immediate 


52  INORGANIC  FOODSTUFFS 

associates"  the  precise  object  of  the  measures  advocated,  their  t 
porary  character,  and  the  fact  that  they  are  applicable  only  to 
particular  case  in  point,  and  not  to  humanity  in  general,  irrespeci 
of  age,  sex,  health  or  disease. 

We  will  take  up  the  question  of  the  dietary  requirements  of  the  b< 
in  several  subsequent  chapters  and  in  a  variety  of  connections.     T 
above  remarks  will,  however,  be  found  to  apply  only  the  more  forcit ! 
with  the  expansion  of  our  acquaintance  with  the  complexity  and  vark 
of  the  problems  of  nutrition. 


REFERENCES. 
GENERAL: 

Albu-Neuberg:    Physiologic  und  Pathologie  des  Mineralstoffwechsels.     Berlin ,  19 

Osborne  and  Mendel:     Jour.  Biol.  Chem.,  1918,  34,  p.  131;  1913,  15,  p.  311.     C. 
negie  Inst.  of  Washington,  Pub.  156,  1911,  Pt.  II. 

McCollum  and  Davis:     Jour.  Biol.  Chem.,  1913,  14,  p.  xl;  1915,  21,  p.  615. 
SODIUM  AND  POTASSIUM: 

von  Bunge:     Text-Book  of  Physiological  and  Pathological    Chemistry,  trans,   by 

Starling,  E.  A.     Philadelphia,  1902. 
CALCIUM: 

Lunin:     Zeit.  physiol.  Chem.,  1881,  5,  p.  31. 

Hopkins:     Jour.   Physiol.,    1912,  44,  p.   425. 

Stehle:     Jour.  Biol.  Chem.,   1917,  31,  p.  461. 

Givens  and  Mendel:     Ibid.,   1917,\31,  p.  421   (which  see    for  Bibliography). 

Givens,  M.  H.:     Ibid.,  1917,  31,  pp.  435  and  441;  1918,  31,  p.  119;  1918,  35,  p.  241, 
IRON: 

von  Bunge:     Cited  above. 

Abderhalden:     Zeit.  Biol.,  1899,  39,  pp.  113  and  483.     Zeit.  physiol.  Chem.,  1902 
34,  p.  500. 

Macallum,  A.  B.:     Jour.  Physiol.,   1894,  16,  p.  268. 

Lapicque:     Arch.  Physiol.  Norm,  et  Path.,  1895,  7,  p.  280. 

Kunkel:     Pfliiger's  Arch.,  1895,  61,  p.  595. 

Hooper  and  Whipple:     Am.  Jour.  Physiol.,   1917,  45,  p.   573. 

Whipple  and  Hooper:     Ibid.,    1917,  45,   p.   576. 


CHAPTER  III. 
THE  CARBOHYDRATES;  THE  MONOSACCHARIDES. 

GENERAL  CHARACTERISTICS. 

The  Carbohydrates  are  extremely  abundant  in  nature,  and  play  an 
exceedingly  important  part  in  the  life-cycle.  In  vegetable  tissues  they 
are  of  importance,  not  only  as  foodstuffs  and  reserve  materials,  but  also 
as  structural  materials.  For  example,  the  walls  of  plant-cells  are 
usually  composed  of  cellulose,  a  complex  carbohydrate.  In  the  animal 
economy  the  carbohydrates  are  chiefly  of  importance  as  food  and 
reserve-materials  and  afford  a  very  important  source  of  kinetic  energy 
to  our  tissues. 

The  carbohydrates  owe  their  name  to  the  fact  that  all  of  them  contain 

carbon  and  in  all  of  them,  moreover,  the  proportion  of  hydrogen  to 

oxygen  is  the  same  as  it  is  in  water,  namely,  2  to  1.   This  is  not  a  very 

satisfactory  definition  of  the  group,  however,  since  many  substances 

are  known  which  correspond  to  such  a  definition  and  yet  are  most 

distinctly  not  carbohydrates.     In  more  exact  terms  it  may  be  said  that 

carbohydrates  are  aldehyde  and  ketone  derivatives  of  the  polyatomic 

•ilcohols.     The   majority   of   the   naturally   occurring   simple   sugars 

:ontain  six  atoms  of  carbon  and  are  termed  Hexoses,  although  some 

Contain  five  atoms  of  carbon  and  are  termed  Pentoses.    From  the  simple 

ftonosaccharides,  more  complex  sugars,  the  Disaccharides,  are  formed 

>y  the  combination  of  two  molecules  of  monosaccharide   with  the 

Jimination  of  a  molecule  of  water.     More  complex   carbohydrates 

till,  the  starches  and  dextrines,  collectively  termed  the  Polysaccharides, 

,re  derived  from  the  monosaccharides  by  the  combination  of  a  variable 

lumber  of  sugar  molecules,  with  the  elimination  of  a  corresponding 

lumber  of  molecules  of  water. 

It  is  only  within  comparatively  recent  times  that  the  artificial 
ynthesis  of  sugar  has  been  accomplished,  but  within  the  brief  period 
>f  thirty  years  nearly  all  of  the  natural  sugars  have  been  synthesized, 
,nd  the  light  which  the  consequent  accessions  to  our  chemical  knowl- 
dge  have  thrown  upon  the  function  and  transformations  of  the  carbo- 
tydrates  in  living  organisms  is  so  great,  that  today  we  are  in  a  position 
o  interpret  countless  phenomena  which  were  entirely  obscure  before 
hese  discoveries  had  been  made. 

The  first  sugar  to  be  synthesized  was  Glycerose,  which  was  prepared 
>y  Emil  Fischer  in  1890.  This  sugar,  which  contained,  however,  only 
hree  atoms  of  carbon  (formula  (CH2OH)2CO  was  prepared  by  the 
entle  oxidation  of  the  triatomic  alcohol,  glycerol  (C3H5(OH)3.  This 
ynthesis  is  particularly  interesting  because  it  establishes  a  connection 
etween  the  carbohydrates  and  the  fats,  since  all  of  the  naturally 
ccurring  fats  contain  a  glycerol  radical.  From  this  sugar  it  was  found 


54  CARBOHYDRATES— MONOSACCHATMJ2ES -^ 

X  . 

possible  to  prepare  a  sugar  containing  six  atoms  of  carbon  in  the  mole- 
cule, by  the  action  of  dilute  alkali.  At  the  same  time  Fischer  succeeded 
in  synthesizing  a  hexose  (that  is  to  say,  a  six  carbon  atom  sugar)  from 
its  elements,  by  the  polymerization  of  formaldehyde  (HCHO),  in 
accordance  with  the  equation: 

6HCHO    =  C6Hi2O6 

and  this  sugar  was  found  to  be  identical  with  that  which  had  been 
synthesized  from  glycerose. 

Examination  of  this  new  sugar  showed,  however,  that  it  differed  in  a 
very  important  property  from  the  naturally  occurring  hexoses,  fruit- 
sugar,  glucose,  or  mannose.  These  sugars,  when  in  solution,  rotate 
the  plane  of  polarization  of  a  beam  of  polarized  light  to  the  right  or 
to  the  left.  The  synthetic  sugar  did  not  rotate  the  plane  of  polarized 
light,  and  hence  a  special  name  was  given  to  it,  Across. 

The  reason  for  the  optical  inactivity  of  acrose  was  found  to  lie  in  the 
fact  that  it  is  a  mixture  of  equal  parts  of  optical  antipodes,  the  one 
rotating  the  plane  of  polarized  light  to  the  right,  and  the  other  to  the 
left  in  equal  degree.  As  a  matter  of  fact  acrose  can  be  decomposed  by 
appropriate  measures  into  optically  active  constituents,  and  according 
to  the  conditions  which  accompany  the  transformation  we  obtain 
fruit-sugar,  mannose,  or  glucose. 

It  is  a  remarkable  fact  that  nearly  all  natural  products  which  are 
derived  from  living  material  are  possessed  in  some  degree  of  Optical 
Activity.  This  was  at  first  thought  to  be  a  peculiarity  of  substances 
formed  by  living  organisms  and  to  point  to  the  operation  within  living 
tissues  of  some  force  peculiar  to  living  matter.  We  now  understand 
that  the  optical  activity  of  the  constituents  of  living  matter  is  due  to 
the  circumstance  of  their  synthesis  in  the  presence  or  through  the 
agency  of  optically  asymmetric  catalyzers. 

The  exact  conditions  upon  which  this  property  of  optical  activity 
depends  were  first  made  clear  by  Le  Bel  and  Van't  Hoff  in  1874. 
Previously  to  this  Pasteur  had  expressed  the  opinion,  based  upon  his 
fundamental  observations  on  the  differing  crystal-forms  of  the  right- 
handed  and  left-handed  varieties  of  tartaric  acid,  that  the  optical 
activity  of  certain  molecules  must  be  attributable  to  a  certain  degree 
of  asymmetry  of  the  molecule.  This  asymmetry,  in  the  case  of 
carbon  compounds,  Van't  Hoff  was  able  to  trace  to  the  carbon  atom. 
If  we  imagine  the  four  valencies  of  a  carbon  atom  to  be  pointing 
toward  the  four  apices  of  a  tetrahedron,  of  which  the  center  is  the 
carbon  atom,  the  following  arrangements  of  four  different  masses  are 
possible: 


HEXOSES  55 

The  difference  between  these  arrangements  resembles  that  between 
an  image  and  its  reflection  in  a  mirror;  the  diagrams  cannot  be  super- 
imposed upon  one  another  so  that  the  corresponding  parts  will  coincide, 
except  by  inverting  one  of  the  diagrams,  and  thereby  converting  it  into 
the  other,  its  mirror-image.  Now  it  would  appear  that  when  a  carbon 
atom  is  united  by  its  valencies  to  four  different  masses,  either  of  the 
above  arrangements  is  possible,  the  one  yielding  a  dextrorotatory  and 
the  other  a  levorotatory  compound.  An  optically  inactive  body  is 
produced  either  by  a  mixture  of  equal  numbers  of  the  two  forms  of 
molecules  or  by  "internal  racemization,"  i.  e.,  by  the  presence  within 
the  molecule  of  two  equally  active  carbon  atoms  rotating  the  plane  of 
polarized  light  in  opposed  directions. 

This  being  the  case,  the  number  of  possible  optical  isomers  of  a 
substance  which  contains  two  asymmetric  carbon  atoms  is  four,  since 
either  of  the  two  possible  varieties,  levo-  and  dextro-  of  the  first  asym- 
metric atom  may  be  combined  with  either  of  the  two  possible  varieties 
of  the  remaining  atoms.  Similarly  the  number  of  possible  optical 
isomers  of  a  substance  which  contains  three  asymmetric  carbon  atoms 
is  eight,  since  any  of  the  four  possible  arrangements  about  the  first 
two  atoms  may  be  combined  with  either  of  the  two  possible  arrange- 
ments about  the  third  atom,  and,  in  general,  the  number  of  possible 
optical  isomers  of  a  substance  which  contains  n  asymmetric  carbon 
atoms  is  2n. 

THE   HEXOSES. 

The  relationships  which  have  been  described  above  are  very  well 
illustrated  among  the  hexoses.  A  large  number  of  sugars  are  known 
which  possess  the  formula  C6H]2O6.  The  structural  formulae  of  these 
sugars  have  been  elucidated  by  Fischer  and  others,  and  it  has  been 
shown  that  a  number  of  these  possess  a  structure1  which  can  be  repre- 
sented by  the  general  formula: 

CHO 
*CHOH 

*CHOH 

I 
*CHOH 

*CHOH 
CH2OH 

It  will  be  observed  that  the  four  carbon  atoms  which  are  distin- 
guished by  asterisks  are  asymmetric,  because  they  are  each  united 
with  four  different  masses.  For  example,  take  the  second  carbon  atom 
from  the  top  of  the  diagrammatic  formula.  It  is  united  with  the 
following  groups:  -CHO,  -H,  -OH  and  -C4H5(OH)4.  According 
to  the  rule  which  is  enunciated  above,  there  must  be  24  =  16  possible 
optical  isomers  of  this  compound. 

1  Or  are  readily  convertible  into  substances  possessing  such  a  structure,  cf.  below: 


56 


CA  RBOH  YDRA  TES—M  ON  OS  A  CCHA  RIDES 


This  will  be  rendered  clearer  by  the  accompanying  diagram,  which 
illustrates  the  structure  of  the  sixteen  possible  stereo-isomers  of  any 
compound  which  contains  four  asymmetric  carbon  atoms.  Designat- 
ing a  dextrorotatory  carbon  by  the  symbol  +  and  a  levorotatory 
carbon  by  the  symbol  —  it  will  be  seen  that  each  carbon  is  dextrorotatory 
in  eight  isomers,  and  levorotatory  in  eight  others.  It  is  also  evident 
that  provided  the  end-groups  attached  respectively  to  the  first  and 
fourth  asymmetric  carbons  are  identical,  the  isomer  number  11  is 
identical  with  the  isomer  number  5,  12  with  6,  13  with  7  and  so  forth. 


11 


12 


13 


14 


15 


16 


10 


This  is  what  actually  occurs  in  the  corresponding  polyatomic  alcohols,  in 
which  the  —  CHO  group  of  the  sugar  is  replaced  by  the  group  —  CH2OH. 
In  the  hexoses,  of  which  glucose  is  a  representative,  the  two  end- 
groups  are,  of  course,  different  and  hence  no  two  possible  isomers  are 
identical.  There  are,  therefore,  16  possible  sugars  or  hexoses  of  the 
aldehyde  type,  possessing  the  above  formula.  We  may  represent  them 
as  follows,  using  the  prefixes  d-  and  1-  to  signify  dextro-  and  levo- 
rotatory respectively. 


CHO 

I 

H— C— OH 
H— C— OH 
HO— C— H 
HO— C— H 

CH2OH 

1-mannose. 
CHO 

HO— C— H 

I 
H— C— OH 

HO— C— H 
H— C— OH 

s  OHsOH 

1-idosc. 


CHO 

HO— C— H 
HO— C— H 
H— C— OH 
H— C— OH 

CH2OH 

d-mannose. 
CHO 

H— C— OH 

HO— C— H 

I 
H— C— OH 

HO— C— H 

I 

CH2OH 
d-idoso. 


CHO 


HO— C— H 

H— C— OH 
HO— C— H 

HO— C— H 

I 

CH2OH 
1-glucose. 
CHO 


H— C— OH 
H— C— OH 
HO— C— H 
H— C— OH 

CH2OH 

l-galose. 


CHO 

H— C— OH 
HO— C— H 
H— C— OH 
H— C— OH 

CH2OH 
d-glucose. 
CHO 
I 
HO— C— H 

I 
HO— C— H 

I 
H— C— OH 

HO— C— OH 

I 
CH2OH 

d-galose. 


CHO 

I 
HO— C— H 

I 
H— C— OH 

I 
H— C— OH 

HO— C— H 

CH2OH 

1— galactose. 

CHO 
HO— C— H 

HO— C— H 

I 
HO— C— H 

HO— C— H 

CH2OH 

1-allose. 


HEXOSES 

J 

CHO 

CHO 

1 

H—  C—  OH 
1 
HO—  C—  H 

HO—  C—  H 

H—  C—  OH 
1 
H—  C—  OH 

H—  C—  OH 

H—  C—  OH 
CH2OH 

HO—  C—  H 

1 
CH2OH 

d-galactose. 

1-talose. 

CHO 

1 

CHO 

1 

H—  C—  OH 

H—  C—  OH 

H—  C—  OH 

HO—  C—  H 

H—  C—  OH 

HO—  C—  H 

H—  C—  OH 
CH2OH 

HO—  C—  H 
1 
CH2OH 

d-allose. 

1-altrose. 

57 


CHO 

HO—  C—  H 
HO—  C—  H 

HO—  C—  H 

I 
H—  C—  OH 

CH2OH 
d-talose. 

CHO 

HO—  C—  H 

I 
H—  C—  OH 


H—  C—  OH 

1    I 
CH2OH 

d-altrose. 


The  various  sugars  have  been  prepared  synthetically  and  their  con- 
stitutional formulse  confirmed.  Thirteen  of  them  are  laboratory 
products,  and  only  three  of  them  are  met  with  in  nature,  to  wit: 
d-  glucose,  d-  mannose  and  d-  galactose. 

In  addition  to  these  hexoses  of  the  aldehyde  type,  or  Aldoses,  another 
hexose  of  quite  a  different  type  is  of  very  common  occurrence  in  nature, 
namely  fruit-sugar  or  Fructose.  Unlike  all  of  the  hexoses  considered 
above,  fructose  is  a  sugar  of  the  ketone  type,  or  ketose.  Its  structure 
may  be  represented  by  the  formula : 

CH2OH 

CO 

HO— C— H 
H— C— OH 
H— C— OH 

CH2OH 


Fructose  exists  in  a  dextrorotatory  and  a  levorotatory  form,  the 
one  being  the  mirror-image  of  the  other.  We  customarily  distinguish 
between  dextro-  and  levorotatory  forms  by  the  prefixes  employed 
above,  d-  and  1-;  as,  for  example,  d-glucose  and  1-glucose.  The 
form  of  fructose  which  is  represented  in  the  formula  given  is  the 
levorotary  form,  but  it  is,  nevertheless,  termed  d-fructose,  because  of 
its  close  relationship  to  d-glucose,  which  will  be  apparent  on  comparing 
the  two  f ormulse : 


58  CARBOHYDRATES—  MONOSACCHARIDES 

OHO  CH2OH 

|  I 

H— C— OH  CO 

|  I 

HO— C— H  HO— C— H 

H— C— OH  H— C— OH 
|  I 

H— C— OH  H— C— OH 
|  I 

CH2OH  CH2OH 

4-glucose  d-fructose 

The  mirror-image,  which  is  in  reality  dextrorotatory,  is  therefore  termed 
l-fructose.  The  levorotation  of  d-fructose  has  led  to  its  being  very 
generally  designated  levulose,  by  which  name  we  will  hereafter  fre- 
quently refer  to  it. 

Another  ketose  which  occurs  in  nature  is  d-Sorbinose: 


HO 


which  is  formed  when  the  juice  of  the  mountain-ash  is  exposed  to  air, 
by  the  oxidizing  action  of  a  ferment  upon  the  alcohol  sorbitol  which  is 
contained  in  the  juice. 

REACTIONS   OF  THE   CARBOHYDRATES. 

The  aldehyde  and  ketone,  or  potentially1  aldehyde  and  ketone  struc- 
ture of  the  sugars  renders  them  peculiarly  liable  to  oxidation.  Like 
other  aldehydes  and  ketones,  they  reduce  metallic  oxides  in  alkaline 
solution;  thus  they  reduce  cupric  to  cuprous  oxide,  upon  which  fact 
Fehling's  method  of  sugar-estimation  is  based,  and  they  reduce  silver 
salts  in  ammoniacal  solution,  leading  to  the  formation  of  a  silver  mirror. 
Other  reactions  which  are  characteristic  of  the  sugar-group  are  the 
following : 

On  heating  a  solution  of  sugar  in  concentrated  sodium  or  potassium 
hydroxide,  the  liquid  turns  dark  brown  (Moore's  test). 

If  to  about  0.5  c.c.  of  a  dilute  aqueous  solution  of  glucose  are 
added  a  few  drops  of  a  ten  per  cent,  alcoholic  (acetone  free)  solution 
of  cHnaphthol,  and  1  c.c.  of  concentrated  sulphuric  acid  be  cau- 
tiously run  into  the  lower  part  of  the  tube,  so  that  the  lighter  solu- 
tion floats  upon  the  top  of  the  heavy  acid,  the  zone  of  contact  becomes 
reddish  violet  (Molisch's  test).  This  reaction  is  due  to  the  formation 

i  Cf.  below. 


REACTIONS  OF  THE  CARBOHYDRATES 


59 


of  furfurol  from  the  sugar  by  the  concentrated  acid.  The  furfurol 
then  reacts  with  the  ct-naphthol  yielding  a  colored  product. 

If  sugar  be  heated  to  a  considerable  degree  the  mass  partially 
carbonizes  and  turns  deep  brown.  Numerous  products  are  formed  to 
which  the  collective  name  of  caramel  is  given.  Caramel  has  distinc- 
tively colloidal  properties  and  very  high  coloring-power,  upon  which 
depends  its  use  in  the  artificial  coloring  of  beverages. 

The  sugars  themselves  are  very  soluble  and  on  that  account  are 
frequently  difficult  to  characterize  and  to  purify.  They  form,  how- 
ever, insoluble  or  sparingly  soluble  compounds  with  Phenylhydrazine, 
which  are  of  great  service  in  characterizing  the  various  sugars,  enabling 
us  to  identify  them  in  many  cases  with  a  considerable  degree  of  cer- 
tainty. If  an  aldose,  or  sugar,  that  is,  of  the  aldehyde  type,  be  acted 
upon  by  phenylhydrazine  in  the  presence  of  excess  of  acetic  acid,  the 
following  reaction  occurs: 


CHO 

CH(OH) 
CH(OH) 

I 

CH(OH) 
CH(OH) 
2(OH) 


C6H6NH.NH2 


CH:N.NHC6H6 

I 
CH(OH) 

=  CH(OH) 
CH(OH) 
CH(OH) 

CH2(OH) 

Phenylhydrazone. 


H2O 


The  Phenylhydrazones  are,  for  the  most  part,  readily  soluble;  so 
that  this  stage  of  the  reaction  is  easily  overlooked,  since  the -subse- 
quent secondary  reactions  which  are  about  to  be  described  produce 
sparingly  soluble  substances.  Mannose  is,  however,  an  exception  to 
this  rule,  the  phenylhydrazone  being  very  sparingly  soluble  and  readily 
detected  and  isolated.  Other  hydrazines  such  as  methylphenyl- 
hydrazine,  benzylphenylhydrazine,  and  diphenylhydrazines  also  react 
with  sugars  to  form  hydrazones  which  are  in  some  cases  sparingly 
soluble  and  can  readily  be  separated  and  purified  by  repeated  recrystal- 
lization,  and  identified  by  their  melting-points. 

If  excess  of  phenyl  hydrazine  be  employed,  the  remainder  of  the 
reagent  which  is  not  used  up  in  converting  the  sugar  into  a  hydrazone 
acts  as  an  oxidizing  agent,  converting  a  —  CH(OH)  group  into  a  —CO 
group,  thus: 


CH:N.NHC6H6 

I 

CH(OH) 

CH(OH) 
CH(OH) 
CH(OH) 

CH2OH 

Hydrazone       + 


CH:N.NHC6H6 

I 
CO 

CH(OH) 

I 
+  C6H5NH.NH2   =  CH(OH) 

CH(OH) 

CH2OH 

phenylhydrazine    =  oxidation  product 


C6H6NH2  +  NHs 


aniline   +  ammonia. 


60 


CARBOHYDRATES— MONOSACCHARIDES 


This  oxidation-product  subsequently  reacts  with  yet  another  mole- 
cule of  phenylhydrazine,  with  the  formation  of  an  Osazone: 


CH:N.NHC6H5 

CH:N.NHC6H5 

I 

1 

CO 

C:N.NHC6H5 

1 

1 

CH(OH) 

CH(OH) 

1 

1 

CH(OH) 

+    C6H5NH.NH?    =  CH(OH)                +  H2O 

I 

1 

CH(OH) 

CH(OH) 

i 

CH2OH 

CH2OH 

oxidation-produc  t 

+  phenylhydrazine  =  osazone          +         water 

Glucose,  mannose  and  fructose  all  yield  the  same  osazone.     In  the 

case  of  fructose  the 

reactions  described  above  simply  take  place  in  the 

reverse  order,  thus: 

CH2(OH) 

CH2(OH) 

1 

1 

CO 

C  :N.NHC6H6 

1 

1 

CH(OH) 

CH(OH) 

1 

1 

CH(OH)       + 

C6H5NH.NH2       =      CH(OH                 +  H2O 

.1 

1 

CH(OH) 

CH(OH) 

1 

I 

CH2(OH) 

CH2(OH) 

ketose       + 

phenylhydrazine      =     hydrazone        +       water 

CH2OH 

i 

CHO 

i 

C:N.NHC6H6 

C:N.NHC6H6 

1 

I 

CH(OH) 

CH(OH) 

1 

I 

CH(OH) 

+  C6H6NH.NH2    =  CH(OH)                +  C6H5NH2   +  NH3 

1 

| 

CH(OH) 

CH(OH) 

I 

| 

CH2(OH) 

CH2(OH) 

hydrazone     + 

phenylhydrazine  =       oxidation-product  +  anilin    +  ammonia 

CHO 

CH:N.NHC6H6 

1 

| 

C:N.NHC6H6 

C:N.NHC6H6 

I 

| 

CH(OH) 

CH(OH) 

\ 

| 

CH(OH) 

+  C6H5NH.NH2    =  CH(OH)                +  H2O 

1 

| 

CH(OH) 

CH(OH) 

1 

I 

CH2(OH) 

CH2(OH) 

oxidation-product    + 

phenylhydrazine     =    osazone        +       water 

REACTIONS  OF  THE  CARBOHYDRATES 


61 


The  osazones  as  a  class  are  characterized  by  their  relatively  slight 
solubility  in  water.  They  form  yellow  needle-shaped  crystals  and  the 
shapes  of  the  individual  crystals  and  the  way  in  which  they  group 


c. 

FIG.  1. — Osazone   crystals.     A,  phenylglucosazone;   B,   phenylmaltosazone ;    C,  phenyl- 
lactosazone.     (After  Halliburton.) 

together  are  to  some  extent  characteristic  for  each  osazone.  The 
melting-points  of  the  osazones  are  not  very  definite,  depending  some- 
vhat  upon  the  mode  in  which  the  heat  is  applied,  but  they  are,  as  a 
••ule,  sufficiently  definite  to  serve  as  a  means  of  identification. 


62  CARBOHYDRATES— MONOSACCHARIDES 

When  the  Hexoses  are  heated  for  prolonged  periods  with  dilute 
mineral  acids  (excepting  nitric  acid)  they  yield  Levulinic  Acid  (acetyl 
propionic  acid)  and  formic  acid,  besides  "humin  substances"  containing 
a  higher  proportion  of  carbon  than  the  carbohydrates.  The  reaction 
producing  levulinic  acid  proceeds  as  follows: 

C6Hi2O6      =     CH3CO.CH2.CH2.COOH     +     H.COOH     +     H2O 
hexose        =  levulinic  acid  +     formic  acid     +     water. 

When  nitric  acid  is  employed  Saccharic  Acid  is  produced  (see  below) . 

Levulinic  acid  is  soluble  in  water,  alcohol  and  ether  and  forms  a 
colorless  viscous  liquid  which  boils  at  250°  C.  and  yields  with  silver 
nitrate  a  crystalline  salt  with  the  formula  CH3.CO.(CH2)2COOAg. 

The  Pentoses  do  not  yield  levulinic  acid  on  boiling  with  mineral  acids ; 
on  the  contrary,  they  yield  Furfurol 

HC— CH 

II       II 

HC     C.CHO 

\   / 

o 

which  may  be  collected  by  distillation  and  detected  by  the  aid  of 
aniline  acetate  paper,  which  is  colored  red  by  furfurol.  This  reaction 
may  also  be  used  for  the  estimation  of  pentose  since  the  yield  of  fur- 
furol is  quantitative.  For  this  purpose  the  furfurol  is  distilled  and 
bisulphite  added  to  the  distillate,  when  the  usual  bisulphite  compound 
with  aldehydes  is  formed  and  the  unconsumed  bisulphite  is  estimated 
by  titration  with  iodine.  Or  the  furfurol  may  be  converted  into  the 
phloroglucide  by  addition  of  phloroglucin  and  the  yield  of  this  com- 
pound determined  gravimetrically.  It  should  be  very  carefully  borne 
in  mind,  however,  that  Glucuronic  Acid  and  its  compounds  (see  below) 
also  yield  furfurol  on  treatment  with  dilute  acids  so  that  before  deciding 
that  pentoses  are  present  their  identity  should  be  established  by  the 
formation  of  osazones  and  the  absence  of  glucuronic  acid. 

The  pentoses  also  yield  the  following  reactions: 

The  Orcin  Reaction. — To  a  small  amount  of  the  solution  is  added  an 
equal  volume  of  a  solution  of  orcin  in  concentrated  hydrochloric  acid. 
On  heating,  the  solution  turns  reddish-blue  and  then  bluish-green.  If 
pentoses  are  present  in  abundance,  a  green  precipitate  will  be  obtained 
on  cooling  and  standing.  On  shaking  up  the  mixture  with  amyl 
alcohol  the  green  coloration  passes  over  into  this  solvent.  This 
reaction  is  also  yielded  by  glucuronic  acid. 

The  Phloroglucin  Reaction. — This  reaction  is  carried  out  in  the  same 
manner  as  the  above,  phloroglucin  being  used  in  the  place  of  orcin. 
The  mixture  turns  red  on  heating  and  becomes  cloudy  on  cooling  On 
shaking  with  amyl  alcohol  the  red  color  passes  over  into  the  amyl. 
alcohol  layer.  This  reaction  is  also  yielded  by  glucuronic  acid. 

Selivanoff's  Reaction. — The  Ketoses  may  be  distinguished  from  the 
Aldoses  by  Selivanoff's  reaction,  as  follows: 


REACTIONS  OF  THE  CARBOHYDRATES  63 

To  a  few  cubic  centimeters  of  solution  is  added  an  equal  volume  of 
twenty  per  cent,  solution  of  hydrochloric  acid  containing  a  small  pro- 
portion of  resorcinol.  The  liquid  turns  red  on  heating  and  a  red  sub- 
stance is  gradually  deposited  which  is  soluble  in  alcohol.  This  reaction 
depends  upon  the  formation  of  oxymethyl-f urf urol  from  the  ketose  by 
heating  with  acids.  If  the  acid  be  too  concentrated  or  the  mixture 
boiled  for  more  than  about  twenty  seconds,  the  hexoses  will  also  yield 
a  small  proportion  of  oxymethyl-f  urf  urol  and  will  in  consequence  yield 
the  same  reaction.  It  is  necessary  therefore  to  avoid  a  higher  con- 
centration of  hydrochloric  acid  in  the  final  mixture  than  about  twelve 
per  cent.,  and  to  boil  only  for  a  period  of  less  than  twenty  seconds. 
This  danger  of  confusion  with  the  aldoses  may  be  avoided  by  employing 
glacial  acetic  acid  containing  a  small  proportion  of  hydrochloric  acid, 
instead  of  concentrated  hydrochloric  acid,  as  the  solvent  for  the 
resorcinol. 

The  Chemical  Relationships  of  the  Sugars. — Each  sugar  being  an 
aldehyde  or  ketose,  or  at  any  rate  potentially  an  aldehyde  or  ketose, 
corresponds  to  an  alcohol  from  which  it  is  derived  by  oxidation.  Re- 
duction of  the  sugars,  therefore,  results  in  the  formation  of  alcohols. 
Glucose  yields  Sorbitol,  mannose  yields  Mannitol  and  galactose  yields 
Dulcitol.  The  following  are  the  formulae  which  illustrate  the  structure 
of  these  alcohols  and  their  derivation  from  the  corresponding  sugars. 

CHO  CH2OH  CHO  CH2OH 

I  I  I  I 

HC— OH  H— C— OH  HO— C— H  HO— C— H 

I  II  I 

HO— C— H  HO— C— H  HO— C— H  HO— C— H 

+  H2  =  +  H2   =  | 

H— C— OH  H— C— OH  H— C— OH  H— C— OH 

1  II  I 

H— C— OH  H— C— OH  H— C— OH  H— C— OH 

I  II  I 

CH2OH  CH2OH  CH2OH  CH2OH 

d-glucose.  d-sorbitol.  d-mannose.  d-mannitol. 

CHO  CH2OH 

H— C— OH  H— C— OH 

HO— C— H  HO— C— H 

I  +  H2   =  | 

HO— C— H  HO— C— H 

H— C— OH  H— C— OH 

CH2OH  CH2OH! 

d-galactose.  d-dulcitol. 

All  of  these  alcohols  occur  in  plants,  mannitol  especially  being  widely 
distributed.  They  have  a  sweet  taste,  but  they  are  not  fermentable 
by  yeast. 

Just  as  the  sugars,  being  potential  aldehydes  or  .ketoses,  are  con- 


64 


CARBOHYDRA  TES—MONOSACCHARIDES 


verted  by  reduction  into  Alcohols,  so,  by  oxidation,  they  are  converted 
into  acids.  Glucose  yields  three  different  6-carbon  atom  acids  on 
oxidation.  Two  of  these  acids  are  monobasic  and  the  third  is  dibasic. 


CH2OH 

Glucose. 


COOH 
I 
H— C— OH 

HO— C— H 
H— C— OH 
H— C— OH 

CH2OH 

Gluconic  acid. 


CHO 

H— C— OH 
HO— C— H 
H— C— OH 

H— C— OH 

I 

COOH 
Glucuronic  acid. 


COOH 
I 
H— C— OH 

HO— C— H 

I 
H— C— OH 

I 
H— C— OH 

I 

COOH 
Saccharic  acid. 


From  mannose  the  dibasic  acid  which  is  obtained  is  Manno-sac- 
charic  Acid.  From  galactose  the  dibasic  acid  which  results  from 
oxidation  is  Mucic  Acid.  The  ketoses,  including  levulose,  behave  quite 
differently  on  oxidation.  The  aldoses,  on  being  oxidized  yield  acids 
containing  the  same  number  of  carbon  atoms  as  the  original  sugar. 
The  ketoses,  on  the  contrary,  break  down  on  oxidation  and  yield 
compounds  containing  fewer  carbon  atoms  than  the  original  sugar. 

Similar  relationships  subsist  among  the  sugars  which  contain  fewer 
than  six  carbon  atoms.  Thus  we  have: 


BIOSES. 


Alcohol. 
CH2OH 

I 
CH2OH 

Glycol. 


Sugar. 
CHO 

CH2OH 

Glycolose. 


Monobasic  acid. 
COOH 
I 

CH2OH 
Gly collie  acid. 


Dibasic  acid. 
COOH 

COOH 

Oxalic  acid. 


TRIOSES. 


Alcohol. 
CH2OH 

CHOH 

CH2OH 
Glycerol. 


CH2OH 
I 
CHOH 

CHOH 

CH2OH 

Erythritol. 


Sugar. 
CHO 

CHOH 

CH2OH 

Glycerose. 


Monobasic  acid. 
COOH 

CHOH 

CH2OH 

Glyceric  acid. 


TETROSES. 
CHO  COOH 


CHOH 

I 
CHOH 

I 

CH2OH 
Erythrose. 


CHOH 
CHOH 

CH2OH 

Erythric  acid. 


Dibasic  acid. 
COOH 


CHOH 

I 
COOH 

Tartronic  acid. 


COOH 
I 
CHOH 

CHOH 

COOH 

Tartaric  acid. 


Similarly,  the  pentose  Arabinose  corresponds  to  the  alcohol 
Arabitol  and  to  the  acids  Araboric  and  Trioxyglutaric,  while  the  pentose 
Xylose  corresponds  to  the  alcohol  Xylitol. 


OF   THE  CARBOHYDRATES  •        65 


By  appropriate  methods  it  is  possible  to  convert  sugars  into  others 
containing  more  carbon  atoms  and  vice  versa.  Thus  the  aldoses 
combine  directly,  with  Hydrocyanic  Acid  with  the  formation  of  Nitriles, 
in  accordance  with  the  equation: 

C5Hn05.CHO     +     HCN     =     C6HuO6CH(OH).CN 

These  nitriles^on  hydrolysis,  yield  acids  containing  one  carbon  atom 

more  than  the  original  carbohydrates,  thus: 

C5HnO5.CH(OH).CN     +     2H2O      =     C5HUO5CH(OH)COOH     +     NH3 

Reduction  of  these  acids,  by  means  of  sodium  amalgam,  yields  the 
corresponding  aldose  with  one  carbon  atom  more  than  the  original 
sugar.  In  this  way  glucose  has  been  prepared  from  arabinose,  and 
seven-  and  even  nine-atom  sugars  have  also  been  prepared,  by  successive 
steps,  starting  with  glucose. 

The  conversion  of  a  sugar  containing  more,  into  one  containing  fewer 
carbon  atoms  may  be  accomplished  by  converting  the  sugar  by  gentle 
oxidation  into  the  corresponding  (monobasic)  acid,  and  then  subjecting 
the  calcium  salt  of  this  acid  to  further  oxidation,  with  the  result  that 
the  carboxyl-group  is  decomposed  into  carbon  dioxide  and  water,  and 
a  sugar  containing  one  less  carbon  atom  than  the  original  sugar  is 
formed : 


CHO  COOH 

I  i 

CH(OH)  CH(OH)  A    COH 

I  I  if  I 

CH(OH)  CH(OH)  CH(OH) 

I  I  t/l 

CH(OH)  CH(OH)  -  CH(OH)     +  CO2  +  H2O 

I  I  .1 

CH(OH)  CH(OH)  CH(OH) 

i  I  ! 

CH2(OH)  CH2(OH)  CH2(OH) 

Aldo-hexose  Aldonic  acid  Aldo-pentose 

This  reaction  is  of  very  great  interest  to  the  biochemist  because  the 
conversion  of  a  carboxyl-  group  into  CO2  and  H20  is  known  to  be  readily 
accomplished  by  bacterial  action  and  probably  also  by  animal  tissues. 
TJie  possibility  is  thus  indicated  that  pentoses  in  the  tissues  may  be 
derivable  from  glucose,  a  possibility,  the  significance  of  which  will  be 
apparent  at  a  later  stage. 

Not  only  is  it  possible  to  convert  a  hexose  into  a  pentose  and  vice 
versa,  but  it  is  also  possible  to  convert  one  hexose  into  another.  It  was 
found  by  Lobry  de  Bruyn  that  in  the  presence  of  alkalies,  glucose, 
mannose  or  levulose  in  aqueous  solution  yields  a  mixture  of  the  three 
sugars.  More  concentrated  alkali  brings  about  more  pronounced 
decomposition,  as  is  evidenced  by  the  formation  of  lactic  acid  and  other 
hydroxy-acids  in  Moore's  test  for  carbohydrates.  The  production  of 
lactic  acid  from  glucose  by  the  action  of  alkalies  is  a  phenomenon  of 
5 


MONOS^^f. 


66  CARBOHYDRATES— MONOSAlTTIARIDES 

great  importance  in  the  light  of  the  fact  that  the  decomposition  of 
glucose,  or  glycogen  which  is  an  anhydride  of  glucose,  in  muscular 
tissue  leads  to  the  formation  of  lactic  acid. 

The  action  of  alkalies  upon  glucose  led  to  the  suspicion  that  if  am- 
monia were  employed  amino-derivatives  of  hydroxy-acids,  such  as  are 
found  among  the  constituent  radicals  of  the  proteins,  might  possibly 
be  formed,  and  it  was  found  by  Windaus  and  Knoop  that,  as  a  matter 
of  fact,  ammonia,  acting  upon  glucose,  mannosiMJtalose,  sorbose, 
arabinose,  xylose,  rhamnose  or  lactose  yields  Methyl^^oxaline : 

CH3C— NH 

>CH 

HC— N/ 

No  other  amino-products  of  this  decomposition  were  identified,  but 
this  one  is  of  extraordinary  interest,  because  of  the  very  great  impor- 
tance and  variety  of  roles  played  by  the  Iminazole  ring  in  physiological 


phenomena.  While  it  is  very  doubtful  whether  the  synthesis  of  this 
ring  is  possible  for  animal  tissues  to  accomplish,  and  in  fact  there  is 
much  evidence  tending  to  show  that  it  is  not,  yet  it  is,  of  course,  unques- 
tionably accomplished  by  vegetable  tissues,  since  the  iminazole  ring 
in  the  form  of  the  amino-acid  Histidine  and  in  the  purine-base  moiety 
of  the  Nucleic  Acids,  is  an  invariable  and  essential  constituent  of  living 
matter. 

On  heating  hexoses  in  concentrated  solution  with  amino-acids  (glyco- 
coll,  alanine,  leucine,  tyrosine  or  glutamic  acid)  the  mixture  darkens 
with  the  formation  of  "Humin  Substances"  which  are  very  deeply 
colored.  At  the  same  time  carbon  dioxide  is  discharged  from  the 
mixture.  It  is  believed  that  the  carbon  dioxide  is  released  from  the 
carboxyl-group  of  the  amino-acid  which  unites  with  the  aldehyde- 
group  of  the  sugar  to  form  cyclic  compounds.  Similar  substances  are 
formed  (from  tryptophane)  when  proteins  are  hydrolyzed  by  strong 
acids  in  the  presence  of  carbohydrates. 

Certain  Derivatives  of  Glucose. — Two  derivatives  of  glucose, 
Glucuronic  Acid  and  Glucosamin,  claim  our  attention  at  this  juncture, 
because  they  are  both  of  profound  physiological  importance. 

We  have  seen  that  on  oxidation,  glucose  yields,  first  two  monobasic 
acids  and  thereafter,  on  continued  oxidation,  each  of  these  monobasic 
acids  yields  the  same  dibasic  acid.  One  of  the  monobasic  acids  is 
glucuronic  acid,  the  dibasic  acid  is  saccharic  acid.  The  connection 
between  glucose,  glucuronic  acid  and  saccharic  acid  can  be  seen  at  a 
glance  from  their  formulae: 


REACTIONS  OF  THE  CARBOHYDRATES         67 

CHO  CHO  COOH 

I  I  I 

H— C— OH  H— C— OH  H— C— OH 

I  I  I 

HO— C— H  HO— C— H  HO— C— H 

H— C— OH  H— C— OH  H— C— OH 

H— C— OH  H— C— OH  H— C— OH 

I  I  I 

CH2OH  COOH  COOH 

d-glucose.  d-glucuronic  acid.  d-saccharic  acid. 

Glucuronic  acid  is  therefore  at  the  same  time  an  acid  and  an  alde- 
hyde. On  boiling  its  solution  or  on  prolonged  standing  it  is  trans- 
formed into  a  crystalline  lactone  which  is  represented  by  the  formula: 

CHO 
H— c— OH 


Glucuronic  acid  yields  the  pentose  reactions  with  orcin  or  phloro- 
glucin  and  hydrochloric  acid  (see  p.  62)  and  also  the  following  reaction: 

Naphtho-resorcinol  Reaction. — A  small  amount  of  naphtho-resorcinol 
is  dissolved  in  concentrated  hydrochloric  acid  and  to  this  reagent  is 
added  an  equal  volume  of  a  solution  of  glucuronic  acid.  A  violet-blue 
coloration  results  which  is  soluble  in  ether.  This  reaction  is  not  specific 
for  glucuronic  acid,  being  given  by  many  ketose  and  aldehyde  acids. 
It  is,  however,  useful  for  the  purpose  of  distinguishing  between  glu- 
curonic acid  and  the  pentoses. 

Glucuronic  acid  does  not  occur  in  the  free  condition  in  animal  tissues, 
nor  has  it  as  yet  been  identified  in  plants.  In  the  form  of  ester-like 
compounds,  however,  it  is  found  in  many  plants,  notably  in  Scutellaria, 
and  esters  of  glucuronic  acid  are  found  in^many  parts  of  the  body, 
in  the  blood,  the  liver  and  in  urine.  The  normal  forms  in  which  it  is 
found  in  urine  are  Phenyl-,  Indoxyl-  and  Skatoxyl-glucuronic  acids. 
Indoxyl  and  skatoxyl  are  highly  toxic  products  of  intestinal  putrefac- 
tion; the  compounds  which  they  form  with  glucuronic  acid  are,  however, 
harmless. 

Under  ordinary  conditions,  glucose  is  readily  oxidized  in  the  body  to 
carbon  dioxide  and  water,  passing  through  intermediate  stages  of  which 
lactic  acid  is  one.  But  in  the  presence  of  some  toxic  agents  it  appears 
that  the  oxidation  of  glucose  is  arrested  at  the  formation  of  glucuronic 
acid,  which  combines  with  the  toxic  substance,  the  compound  being 


68  CA  RBOH  YDRA  TES—MONOSA  CCHARIDES 

eliminated  as  such.  It  is  not  known  whether  or  not  glucuronic  acid 
is  a  normal  intermediate  product  of  glucose  oxidation  in  the  animal 
body,  or  whether  it  is  only  formed  under  the  exceptional  condition  of 
the  presence  of  certain  toxic  bodies.  Large  quantities  of  glucuronic 
acid,  in  these  ester-like  combinations,  appear  in  the  urine  when  certain 
drugs  are  introduced  into  the  system.  The  following  is  a  partial  list 
of  the  drugs  which  are  eliminated  in  this  manner : 

Isopropyl  alcohol  chloral 

Methyl  propyl  alcohol  butyl  chloral 

Methyl  ethyl  carbinol  bromal 

Tertiary  butyl  alcohol  dichloracetone 

Tertiary  amyl  alcohol  a  and  /3  napthol 

Benzol  turpentine 

Nitrobenzol  camphor 

Aniline  borneol 

Phenol  menthol 

Resorcinol  pinene 

Thymol  antipyrine 

The  elimination  of  these  drugs  in  this  manner  constitutes  a  pitfall 
for  the  unwary  who  may  seek,  after  the  administration  of  such  drugs 
as  these  to  a  patient,  to  investigate  the  urine  for  the  presence  of  sugar 
therein  by  the  phenylhydrazine  test,  or  its  clinical  modification  known 
as  Cipollina's  test.  For  glucuronic  acid  forms  an  osazone  which  may 
easily  be  mistaken  for  glucosazone.  The  distinction  may  very  readily 
be  made,  however;  owing  to  the  fact  that  the  osazone  of  glucuronic 
acid  is  decomposed  by  heating  while  that  of  glucose  is  not.  If  the 
precaution  be  taken,  therefore,  of  heating  the  precipitate  on  a  boiling 
water-bath  for  half  an  hour  before  examining  it,  no  confusion  of  the 
osazone  of  glucuronic  acid  with  glucosazone  is  possible,  for  the  osazone 
of  glucuronic  acid  is  decomposed  by  this  procedure  and  redissolves, 
while  the  osazone  of  glucose  remains  unaltered. 

Glucuronic  acid  is  therefore  to  be  regarded  as  a  protective  agent 
which  guards  the  organism  against  the  deleterious  action  of  certain 
substances  introduced  from  without  or,  in  some  cases,  from  within  the 
body.  Sometimes  the  glucuronic  acid  accomplishes  its  protective 
function  by  combining  directly  with  the  toxic  substance,  rendering  it 
harmless  until  in  the  course  of  time  it  is  eliminated;  in  other  instances 
the  toxic  substance  unde^oes  some  degree  of  change  and  elaboration 
before  it  is  paired  with  glucuronic  acid.  Thus  chloral  hydrate  and 
butyl  chloral  undergo  reduction  before  they  couple  with  the  glucuronic 
acid;  o-nitro toluol,  on  the  contrary,  is  oxidized  to  nitrobenzyl-alcohol 
before  it  pairs  with  glucuronic  acid.  Other  substances  undergo 
hydration  or  both  hydration  and  oxidation  before  they  can  couple  with 
the  glucuronic  acid. 

Glucuronic  acid  is  possibly  of  importane  not  only  as  a  carrier  of 
toxic  substances  out  of  the  body,  but  also  as  a  connecting-link  between 
the  hexoses  and  the  pentoses.  It  will  be  recollected  that  when  a  mono- 


MONOSACCHARIDES  IN  LIVING  TISSUES  69 

basic  acid  derivative  of  an  aldohexose  is  acted  upon  by  oxidizing  agents, 
the  carboxyl  group  is  eliminated  in  the  form  of  carbon  dioxide  and 
water,  and  the  corresponding  aldo-pentose  is  formed.  When  d-glucu- 
ronic  acid  is  subjected  to  intense  putrefaction,  it  undergoes  an  analogous 
change,  with  the  production  of  1-xylose,  thus : 

CHO  CHO 

I  I 

H— C— OH       .         H— C— OH 

I  I 

HO— C— H  HO— C— H 

+      CO2 
H— C— OH  H— C— OH 

H— C— OH  CH2OH 

COOH 

Glucosamin,  on  the  other  hand,  affords  a  connecting  link  between 
the  carbohydrates  and  the  hydroxy-amino-acids.  It  is  readily  obtained 
in  considerable  quantities  from  the  exoskeletons  of  Crustacea,  as  for 
example  from  the  shells  of  lobsters,  by  boiling  with  concentrated 
hydrochloric  acid.  It  also  occurs  in  fungi  and  it  is  a  constituent  of  the 
Mucins  and  Mucoids;  sticky  glutinous  proteins  which  are  found  in 
mucous  secretions  and  elsewhere.  The  formula  of  glucosamin  is: 

CHO 

H— C— NH2 
HO— C— H 
H— C— OH 
H— C— OH 
CH2OH 

In  the  true  mucins,  but  not  in  the  mucoids  the  glucosamin  radical  is 
probably  acetylated,  and  acetyl  glucosamin,  in  common  with  other 
acetyl  derivatives  of  hydroxy-amino-acids,  yields  Ehrlich's  Reaction, 
namely  a  pink  color  when  its  solution  is  mixed  and  warmed  or  allowed 
to  stand  with  an  equal  volume  of  a  two  per  cent,  solution  of  paradi- 
methylaminobenzaldehyde  in  hydrochloric  acid  of  specific  gravity 
1.09.  The  mucins  also  yield  this  reaction. 


THE  DISTRIBUTION  OF  THE  MONOSACCHARIDES  IN  LIVING 

TISSUES. 

As  has  been  stated,  a  pentose,  d-Ribose  is  a  normal  constituent  of 
the  nucleoproteins.     The  following,  after  Grund,  is  the  percentage 


70  CARBOHYDRATES— MONOSACCHARIDES 

of  pentoses  calculated  on  the  basis  of  the  dry  tissue,  which  is  present 
in  various  parts  of  the  mammalian  body: 

Pancreas 2.48 

Liver 0.56 

Thymus 0.56 

Submaxillary  gland 0.53 

Thyroid  gland 0.50 

Kidneys 0.49 

Spleen 0.46 

Brain 0.22 

Muscle 0.11 

The  structural  formula  of  d-ribose  may  be  represented  as: 

CHO 

I 

H— C— OH 

I 
H— C— OH 

H— C— OH 

CH2OH 

d-ribose. 

it  is  levorotatory,  the  prefix  d-  being  employed  to  denote  its 
relationship  to  d-altose  and  d-altrose. 

In  certain  very  exceptional  cases  a  pentose  is  formed  in  the  urine. 
The  disease  which  leads  to  this  elimination  of  pentoses  is  known  as 
Pentosuria,  in  contradistinction  to  Glycosuria,  the  very  much  more 
common  elimination  of  -glucose.  Only  a  few  cases  of  pentosuria  have 
been  observed,  but  it  is  an  extremely '  noteworthy  fact  '  that  the 
pentose  which  is  eliminated  in  this  disease  would  appear  to  be  almost 
invariably  optically  inactive,  although  the  pentose,  1-ribose,  which  is 
normally  found  in  the  tissues  is,  of  course,  optically  active.  Not  only 
this,  but  the  pentose  in  the  urine  is  not  ribose  but  Arabinose, 

CHO  CHO 

I  I 

HO— C— H  H— C— OH 

I  I 

H— C— OH  HO— C— H 

H— C— OH  HO— C— H 

I  I 

CH2OH  CH2OH 

d-arabinose.  1-arabinose. 

which  would  seem  to  point  to  its  derivation  from  glucose  rather  than 
from  the  decomposition  of  nucleo-proteins,  for  it  will  be  remembered 
that  arabinose  may  be  derived  from  glucose  by  the  oxidation  of  the 
calcium  salt  of  gluconic  acid  (xylose  being  the  corresponding  pentose 
resulting  from  the  oxidation  of  glucuronic  acid).  However  this  may 
be,  the  pentose  elimination  in  these  cases  is  independent  of  the  pentose- 


MONOSACCHARIDES  IN  LIVING  TISSUES  7l 

content  of  the  food  and  may  occur  when  the  combustion  of  carbo- 
hydrates in  the  tissues  would  appear  to  be  otherwise  normal. 

The  pentoses  are  widely  distributed  in  the  vegetable  kingdom, 
chiefly  in  the  form  of  polysaccharides,  bearing  the  same  relation  to  the 
pentoses  as  starch  and  glycogen  do  to  the  hexoses.  These  polysac- 
charides, which  are  complex  anhydrides  of  the  monosaccharides,  are 
known  as  Pentosans.  The  following  table  shows  the  percentage  of 
pentosan,  in  terms  of  pentose,  found  in  the  dry  substance  of  various 
vegetable  foods: 

Meadow  hay    .      ,      .      .      .      .      .    '.      .      .      .      .      ...      .      .      .  21.64 

Rape  cake .  •                                       ....  11.50 

Oil-seed  cake ...>....      .  9.07 

Bruised  barley        .      .      .      .      .      .      .      .      .                  .      .      ....  7.96 

Rice  flour ;...-.       ..    »    :.      .      .  5.73 

Sesame  cake .     •.      .      ,.    .      .      .      .  3.87 

Table  turnip     .      ., .      .      .'...'.      .      .      .  1.13 

Spinach .      .      .      .      .  ^    1.02 

With  regard  to  the  distribution  of  the  hexoses;  Levulose  is  not 
often  found  in  the  animal  kingdom.  In  honey  it  occurs  together  with 
glucose  and  is  immediately  derived  from  the  juices  of  flowers,  but  it  is 
a  question  whether  it  is  ever  normally  found  in  animal  tissues.  It  is 
occasionally  found  in  the  urine,  and  is  then  derived  directly  from  the 
levulose  absorbed  from  the  intestine;  it  may  be  regarded  as  a  sign 
either  of  excessive  overindulgence  in  sweets  or  honey  or  else,  if  this 
origin  can  be  excluded,  as  a  sign  of  overactivity  of  the  pituitary  gland, 
which  as  we  shall  see  later  on,  lowers  the  limit  of  tolerance  for  all  forms 
of  sugar.  A  urine  which  yields  evidence  of  the'  presence  of  a  reducing 
sugar  should  therefore  always  be  tested  for  levulose  by  Selivanoff's 
test  (see  p.  62)  before  a  provisional  diagnosis  of  diabetes  is  decided 
upon. 

In  vegetable  tissues  levulose  is  widely  distributed,  especially  com- 
bined with  glucose  to  form  cane-sugar.  It  is  also  found  in  the  form 
of  a  complex  anhydride,  or  polysaccharide,  Inulin  in  the  tubers  of 
dahlias  and  in  the  sweet  potato. 

Grape-sugar,  or  d-glucose,  is  the  most  important  of  all  the  mono- 
saccharides in  the  animal  economy.  It  is  the  central  figure  in  the 
carbohydrate  metabolism.  Polysaccharides  are  broken  down  to 
glucose  before  assimilation,  and  again  before  utilization  as  a  source 
of  energy,  or  transportation  from  one  part  of  the  body  to  another.  It 
is  the  circulating  form  of  carbohydrate,  Glycogen  and  other  poly- 
saccharides being  the  storage-forms.  In  view  of  these  facts  the 
absurdity  will  be  apparent  of  the  effort  which  was  made  in  certain 
circles  in  the  United  States,  a  few  years  ago,  to  represent  glucose  and 
glucose-syrups  as  deleterious  articles  of  food.  Provided  they  contain 
no  other  'constituents  which  are  harmful  such  preparations  are  merely 
solutions  of  the  only  carbohydrate  which  is  to  any  important  extent 
a  normal  and  invariable  constituent  of  the  blood. 


72  CARBOHYDRA  TES—MONOSACCHARIDES 

Normal  urine  contains  minute  traces  of  glucose,  and  sometimes 
larger  amounts,  especially  after  a  meal  which  is  very  rich  in  carbo- 
hydrates. Such  glycosuria  is  known  as  Alimentary  Glycosuria  and  is 
devoid  of  significance  unless  it  occurs  too  frequently  and  readily,  in 
which  case  it  may  possibly  indicate  disturbance  of  the  functions  of  the 
pituitary  gland.  In  certain  pathological  conditions  or  under  experi- 
mental conditions  much  profound  and  serious  glycosurias  may  occur. 
These  will  fall  under  consideration  in  a  later  chapter. 

Galactose  is  found  in  important  quantities  in  two  places  in  the 
animal  kingdom,  namely  combined  with  glucose  to  form  milk-sugar 
or  Lactose;  and  in  the  form  of  glucoside-like  compounds,  the  Cere- 
brosides,  which  are  found  in  the  brain. 


THE  LACTONE-STRUCTURE  OF  SUGARS. 

Before  proceeding  to  the  consideration  of  the  disaccharides,  it  is 
important  to  review  some  recent  accessions  to  our  knowledge  of  the 
sugars  which  have  led  us  to  reconsider  in  some  degree  the  structural 
formulae  by  means  of  which  we  have  hitherto  represented  them.  It  is 
necessary  to  enter  thus  deeply  into  the  subject  of  the  configuration  of 
the  sugar  molecule  because  a  clear  understanding  of  these  questions 
has  already  fundamentally  contributed  to  our  knowledge  of  the  mode 
of  action  of  ferments,  and  is  unquestionably  destined  to  do  so  even  to  a 
greater  degree  than  heretofore. '  In  considering  the  enzymatic  hydrol- 
ysis and  synthesis  of  the  disaccharides  we  shall  have  occasion  to  refer 
very  frequently  to  the  facts  which  are  about  to  be  described. 

It  will  be  recollected  that  in  compounds  containing  only  four 
asymmetric  carbon  atoms,  such  as  we  have  been  assuming  the  hexoses 
to  be,  only  sixteen  stereo-isomers  are  possible.  Now,  as  a  matter  of 
fact,  it  has  long  been  known  to  sugar-chemists  that  the  optical  rotatory 
power  of  solutions  of  d-glucose  is  not  a  constant  quantity.  The 
optical  rotatory  power  of  fresh  solutions  changes  gradually,  sometimes 
increasing,  but  more  usually  falling,  until  a  constant  value  is  ultimately 
reached.  This  constant  value  is  the  same  for  all  glucose  solutions 
which  have  attained  equilibrium,  but  the  initial  rotatory  power  of 
fresh  solution  may  be  as  much  as  twice  as  great  as  the  final  constant 
rotatory  power.  This  phenomenon  is  variously  known  as  Mutaro- 
tation,  Multirotation  and  Birotation. 

Analogous  phenomena  in  other  solutions  are  generally  attributed 
to  the  presence  of  two  or  more  different,  optically  active  substances, 
of  different  rotatory  power  and  convertible  into  one  another.  Adopting 
this  point  of  view,  Emil  Fischer  first  suggested,  in  explanation  of  the 
phenomenon  of  mutarotation,  that  the  glucose  undergoes  hydration 
in  solution,  with  the  formation  of  an  alcohol  of  lower  rotatory  power, 
thus: 


LACTONE-STRUCTURE  OF  SUGARS  73 

CHO  CH2(OH) 

I  I 

CH(OH)  CH(OH) 

CH(OH)  CH(OH) 

+  H20 

CH(OH)  CH(OH) 

CH(OH)  CH(OH) 

CH2(OH)  CH2(OH) 

This  view,  which  never  had  any  experimental  support,  was  rendered 
unnecessary  and  untenable  by  the  discovery  of  the  fact  that  two 
different  forms  of  d-glucose  are  obtainable,  isomers  of  one  another 
but  differing  in  rotatory  power.  The  one  form,  a-d-glucose,  crystallizes 
out  at  ordinary  temperatures  from  seventy  per  cent,  alcohol,  and  has 
a  molecular  rotation  of  («)D  +  110°;  the  other,  /3-d-glucose,  crystal- 
lises out  from  solutions  in  water  at  temperatures  above  98°  C.,  and 
has  a  molecular  rotation  of  («)D  +  19°.  It  appears  that  there  are 
indeed  twostereo-isomeric  forms  of  d-glucose,  which  would  be  impossible 
were  there  onlv  four  asvmmetric  carbon  atoms  in  the  molecule,  as  the 
formula  CHO  -  CH(OH)  -  CH(OH)  -  CH(OH)  -  CH(OH)  -  CH2(OH) 
requires.  The  glucose  molecule  must,  in  fact,  contain  not  less  than 
Five  asymmetrical  carbon  atoms.  This  conclusion,  first  suggested  by 
Simon,  was  verified  by  Armstrong  in  the  following  way: 

Two  methylated  d-glucoses  are  known,  formed  from  glucose  by  the 
replacement  of  a  hydrogen  by  a  methyl  group.  The  structures  of  these 
two  methyl  glucosides  are  believed  to  be  respectively: 

CH3— O— CH  HC— O— CH3 


HCOH  \  HCOH  N 


HOCH  '  /  HOCH 

HC/  HC 

HCOH  H 


U' 

COH 


CH2OH  CH2OH 

a-methyl-d -glucoside  /3-methyl-d-glucoside 

Each  of  these  glucosides  can  be  hydrolyzed  by  an  appropriate 
ferment.  Now  it  is  observed  that  a  glucose  of  high  rotatory  power  is 
produced  in  the  hydrolysis  of  the  a-methyl  glucoside,  while  on  adding 
a  drop  of  ammonia  to  the  solution  the  rotation  rapidly  falls  to  the 
equilibrium-value  of  the  rotatory  power  of  ordinary  glucose.  On  the 
other  hand,  when  the  0-methyl  glucoside  is  hydrolyzed,  a  glucose  of 
low  rotatory  power  is  produced,  and  on  adding  a  drop  of  ammonia  to 


74 


CARBOHYDRATES— MONOSACCHARIDES 


the  solution  the  rotatory  power  rapidly  rises  to  the  equlibrium-value 
of  the  rotatory  power  of  ordinary  glucose. 

From  these  observations  it  appears  that  the  true  formula  for  d- 
glucose  is  either: 

HO— C— H  H— C— OH 


HCOH 


\0 


HOCH 


HC/ 

HCOH 


CH2OH 

a-d-glucose. 


HCOH  \ 
HOCH     / 
HC/ 
HCOH 


CH2OH 

p-d-glucose. 


of  which  the  former  is  the  a  (highly  rotating)  form,  and  the  latter  the 
)8  form  of  low  rotatory  power.  In  solution,  an  equilibrium  is  finally 
attained  between  the  two  forms,  and  the  attainment  of  this  equilibrium 
is  much  accelerated  by  an  alkaline  reaction.  The  rotatory  power  of 
the  pure  a  form  is  («)D  +  110°;  that  of  the  pure  (3  form  («)D  +  19°. 
The  rotatory  power  of  an  equilibrated  solution  of  the  mixed  glucoses 
is  (a)D  +  52.5°.  From  these  figures  it  is  a  simple  sum  in  proportion 
to  calculate  that  in  a  ten  per  cent,  solution  of  glucose,  about  thirty- 
seven  per  cent,  is  of  the  a  form  and  about  sixty-three  of  the  £  form  at 
equilibrium. 

We  see  that  glucose  contains,  therefore,  not  four  but  five  asymmetri- 
cal carbon  atoms,  a  fact  which  is  not  revealed  by  a  study  of  long- 
standing or  equilibrated  solutions  and  was  therefore  very  naturally 
overlooked  in  the  first  attempts  to  attach  a  structural  formula  to 
individual  hexoses.  If  this  be  true  of  the  other  hexoses  as  well,  however, 
then  there  must  exist  not  24  =  16  stereo-isomers  of  glucose,  but  2s  =  32. 
As  a  matter  of  fact,  we  find  that  many  of  the  sugars  exhibit  mutaro- 
tation,  for  instance  d-glucose,  d-galactose,  d-mannose,  d-fructose, 
1-arabinose,  l-xylose,  and  some  of  the  disaccharides.  There  is  little 
room  for  doubt  that  the  structural  formulae  of  each  of  these  sugars 
are  analogous  to  the  formulae  for  glucose  which  are  depicted  above. 

Since  the  hexaldoses  all  give  the  aldehyde  reactions,  that  is,  reduce 
metallic  oxides  in  alkaline  solution,  and  unite  with  phenylhydrazine 
by  means  of  an  aldehyde  group,  we  must  suppose  that  in  the  presence 
of  these  reagents  the  oxide  grouping  is  broken  down  and  the  aldehyde 
group  regained.  This  fact  is  very  readily  understood  if  we  suppose 
that  every  solution  of  glucose  contains  a  trace  of  the  aldehyde  form,  in 
equilibrium  with  the  oxide  forms.  A  reagent  such  as  a  metallic  oxide 
or  phenylhydrazine  reacts  with  the  trace  of  aldehyde  form  and  thus 


LACTONE-STRUCTURE  OF  SUGARS  75 

removes  it  from  the  solution;  the  oxide  form  is  then  no  longer  in 
equilibrium  and  therefore  regenerates  the  aldehyde  form  in  the  process 
of  regaining  the  equilibrium  which  has  been  disturbed.  This  fresh 
supply  of  the  aldehyde  form  in  its  turn  reacts  and  is  removed  from  the 
solution,  and  so  the  process  repeats  itself  until  all  of  the  sugar  is  used 
up.  At  the  same  time  this  view  of  the  structure  of  the  sugar  enables 
us  to  understand  why  it  is  that  although  they  give  most  of  the  aldehyde 
reactions,  yet  they  give  them  much  less  energetically  than  the  typical 
aldehydes. 

We  may  also  ascribe  to  the  same  source  the  fact  that  the  sugars  do 
not  react  in  stoichiometrical  proportions  with  metallic  oxides;  in  the 
proportions,  that  is,  which  would  be  expected  if  a  molecule  of  sugar 
reacted  quantitatively  with  a  molecule  of  metallic  oxide.  We  cannot 
predict,  by  merely  writing  down  chemical  equations,  how  much  of  any 
metallic  oxide  under  given  circumstances  will  be  reduced  by  a  given 
amount  of  sugar.  Instead,  for  every  concentration  of  sugar  employed 
and  for  every  circumstance  of  the  reaction,  we  have  to  estimate  afresh, 
and  by  direct  measurement,  the  reducing  power  of  the  sugar.  These 
measurements  are  commonly  expressed  in  tables  which  denote  the 
relationship  of  reduced  cupric  oxide  (or  other  metallic  oxide)  to  the 
quantity  of  sugar  present  in  the  solution  investigated.  But  such  tables 
are  empirically  established  and  are  therefore  reliable  only  if  the  cir- 
cumstances of  concentration,  reaction,  temperature  and  so  forth  are 
exactly  the  same  as  those  which  prevailed  in  the  estimations  from 
which  the  tables  were  computed. 


REFERENCES. 
GENERAL: 

Armstrong:     The  Simple  Carbohydrates  and  the  Glucosides.     London.     2d  edition. 
Levene  and  Jacobs:     Ber.  d.  d.  Chem.  Ges.,  1910,  43,  p.  3141. 
PENTOSES: 

Levene  and  Jacobs:     Ber.  d.  d.  Chem.  Ges.,  1908,  41,  p.  2703;  1909,  42,   pp.  1198, 

2102  and  3247;  1911,  44,  p.  746. 
Grund:     Zeit.  f.  physiol.  Chem.,   1902,  35,  p.   111. 
Bendix  and  Ebstein:     Zeit.  allg.  Physiol.,  1902,  2,  p.  1. 
Clark,  E.  B.:     Jour.  Biol.  Chem.,  1917,  31,  p.  605. 
PENTOSURIA: 

Garrod,  A.  E.:     Inborn  Errors  of  Metabolism.     Oxford  University  Press,  1909. 
DECOMPOSITION  OF  SUGARS: 

Neuberg,  A.:     Oppenheimer's  Handbuch  der  Biochemie,    Erganzungsband,    1913, 

p.  569. 
Levene  and  Meyer:     Jour.  Biol.  Chem.,  1912,  11,  p.  361;  1912,  12,  p.  265;  1913,  14, 

p.  149;  1913,  15,  p.  65. 
AMINO-SUGARS: 

Levene:     Jour.  Biol.  Chem.,   1917,  31,  p.  609. 


CHAPTER  IV. 

THE  CARBOHYDRATES:  THE  DISACCHARIDES,  POLY- 
SACCHARIDES  AND  GLUCOSIDES. 

THE  DISACCHARIDES. 

The  disaccharides  are  carbohydrates  which  contain  twelve  carbon 
atoms,  and  are  formed  from  two  molecules  of  hexose  with  the  elimi- 
nation of  water  in  accordance  with  the  equation: 

C6Hi2O6     +     C6H12O6      =     Ci2H22On     +     H2O. 

The  majority  of  the  disaccharides  reduce  Fehling's  solution  (i.  e., 
cupric  oxide  in  alkaline  solution),  react  with  phenylhydrazine  to  form 
hydrazones  and  osazones,  and  exhibit  mutarotation  in  solution.  They 
therefore  contain  a  potentially  aldehyde  or  ketone  group  or  groups, 
and  an  oxide  linkage  analogous  to  that  in  glucose.  Certain  of  them 
are  exceptions  to  this  rule,  however,  one  of  the  most  marked  excep- 
tions being  cane-sugar,  which  is  formed  by  the  union  of  one  molecule 
of  glucose  with  one  of  fructose  (levulose),  and  which  does  not  reduce 
Fehling's  solution  nor  react  with  phenylhydrazine,  nor  display  muta- 
rotation in  solution. 

The  disaccharides  are  merely  special  instances  of  a  very  large  group 
of  compounds  which  are  generically  termed  Glucosides,  or  compounds 
of  sugars  with  other  bodies,  the  point  of  union  being  the  aldehyde 
group  of  the  sugar.  A  typical  glucoside,  for  example,  is  Amygdalin, 
found  in  cherry-stones  and  in  almonds,  which  on  hydrolysis  yields 
glucose,  hydrocyanic  acid  and  benzaldehyde.  The  nucleic  acids  are 
glucosides.  Glucosides  which  yield  galactose  on  hydrolysis  are  found 
in  the  tissues  of  the  brain.  The  disaccharides  are  glucosides  in  which 
both  constituents  of  the  molecule  are  sugars. 

The  most  important  disaccharides  from  the  point  of  view  of  animal 
biochemistry  are  cane-sugar  or  Sucrose,  Maltose,  Isomaltose,  Lactose 
and  Isolactose.  All  of  these  excepting  sucrose  contain  one  potentially 
active  aldehyde  group;  that  is,  they  reduce  Fehling's  solution,  form 
osazones  and  exhibit  mutarotation. 

Cane-sugar  is  the  ordinary  sugar  of  commerce  and  occurs  widely 
distributed  in  the  vegetable  kingdom,  where  it  acts  as  a  reserve-material, 
that  is,  as  a  store  of  nutriment  to  be  broken  up  into  utilizable  material 
and  consumed  when  needed.  It  occurs  especially  in  the  sugar-cane, 
in  the  sap  of  certain  palms  and  of  the  sugar  maple,  the  birch  and  the 
carob  tree.  Ripe  fruits  and  many  leaves  contain  considerable  amounts 


DISACCHARIDES  77 

of  this  sugar,  while  one  of  the  most  important  sources  of  sugar  is  the 
root  of  the  sugar-beet,  a  variety  which  has  originated  by  selection  from 
the  common  beet  (Beta  maratima).  Cane-sugar  was  not  known  in 
Europe  until  its  introduction  from  the  tropical  parts  of  Asia  where  the 
sugar-cane  has  been  grown  from  time  immemorial.  The  possibility 
of  extracting  cane-sugar  from  beets  was  not  realized  until  it  was  pointed 
out  by  the  German  chemist  Marggraff  in  1760.  Hence  the  large  con- 
sumption of  sugar  now  obtaining  among  European  peoples  is  a  recently 
acquired  habit.  It  is,  of  course,  of  enormous  nutritive  importance  as 
it  results  in  reducing  by  an  equivalent  amount  the  requirement  of 
starch  and  other  polysaccharides.  It  also  enables  us,  when  sugar  from 
the  cane  is  used,  to  utilize  tropical  areas  for  the  production  of  carbo- 
hydrate foodstuffs  and  set  free  greater  areas  of  the  temperate  regions 
for  the  cultivation  of  polysaccharides  and  proteins  (grains,  meat  and 
dairy  products)  for  which  the  tropical  areas  of  the  world  are  not  suit- 
able. The  consumption  of  sugar  from  the  cane  is  therefore  economically 
preferable  to  the  consumption  of  sugar  from  the  beet. 

Cane-sugar  does  not  reduce  Fehling's  solution  nor  does  it  exhibit 
mutarotation.  It  is  neither  potentially  nor  actually  an  aldehyde  or  a 
ketone.  It  is  very  readily  hydrolyzed  by  acids,  therein  differing 
markedly  from  other  disaccharides,  and  it  yields  on  hydrolysis,  one 
molecule  of  d-Glucose  and  one  of  d-Fructose  (levulose).  It  will  be 
recollected  that  d-fructose  is  levorotatory,  and  the  levorotatory  power 
of  d-fructose  being  greater  than  the  dextrorotatory  power  of  d-glucose, 
the  mixed  products  of  cane-sugar  hydrolysis  are  levorotatory.  Cane- 
sugar,  on  the  contrary,  is  dextrorotatory,  so  that  hydrolysis  of  cane- 
sugar  in  solution  leads  to  a  change  of  optical  rotation  from  right  to 
left.  Hence  the  process  of  the  hydrolysis  of  cane-sugar  is  frequently 
termed  Inversion. 

Cane-sugar  is  built  up  by  the  union  of  a  molecule  of  d-glucose  with 
one  of  d-fructose.  The  question  arises,  however,  from  which  of  the  two 
d-glucoses  is  cane-sugar  derived;  the  a-d-glucose  or  the  /3-d -glucose? 
This  question  is  answerable  in  a  very  simple  way.  It  is  possible  to 
hydrolyse  cane-sugar  very  much  more  rapidly  than  a-d-glucose  can 
undergo  transformation  into  /3-d-glucose  or  vice  versa.  It  will  be 
recollected  that  a-d-glucose  possesses  a  much  higher  dextrorotatory 
power  than  /3-d-glucose.  Now  we  find  that  the  glucose  produced  in  the 
hydrolysis  of  cane-sugar  possesses,  initially,  a  high  rotatory  power. 
On  adding  ammonia,  which  accelerates  the  transformation  of  a-  into 
0-glucose,  the  rotation  due  to  glucose  falls.  Hence  the  glucose  set 
free  in  the  hydrolysis  of  cane-sugar  is  a-glucose,  and  cane-sugar  is 
therefore  to  be  regarded  as  a  derivation  of  a-d-glucose. 

Cane-sugar  does  not  react  with  phenylhydrazine.  It  contains 
eight  hydroxyl  groups,  for  it  forms  an  octa-acetate,  in  which  these 
groups  have  been  replaced  by  acetyl  groups.  Apart  from  this  it  has 
not  proved  possible  to  ascribe  any  satisfactory  constitutional  formula 
to  cane-sugar.  The  synthesis  of  cane-sugar  has,  however,  been  accom- 


78    DISACCHARIDES,  POLYSACCHARIDES  AND  GLUCOSIDES 

plished,  by  the  interaction  of  potassium  fructosate  and  acetochlor- 
glucose.1 

Cane-sugar  is  not  attacked  by  any  ferments  excepting  Invertase, 
an  enzyme  found  in  many  yeasts,  moulds,  and  in  some  of  the  higher 
plants.  Invertase,  as  its  name  implies;  effects  the  hydrolysis  of 
cane-sugar  into  its  constituent  parts,  glucose  and  fructose;  that  is, 
it  brings  about  "inversion."  Cane-sugar,  or,  rather,  its  product, 
glucose,  does  not  undergo  alcoholic  fermentation  in  the  presence  of 
yeasts  until  it  is  broken  down  into  glucose  and  fructose.  Hence  yeasts 
which  do  not  contain  invertase  are  not  able  to  cause  alcoholic  fer- 
mentation in  solutions  of  cane-sugar. 

Maltose  is  a  disaccharide  which  results  from  the  hydrolysis  of  starch 
or  of  glycogen  by  acids  or  by  ferments.  Acids,  however,  continue  the 
process  of  hydrolysis  by  splitting  the  maltose  itself,  so  that  maltose 
is  only  a  transient  stage  in  the  hydrolysis  of  starch  or  glycogen  by  acids. 
On  the  other  hand  the  ferments  which  split  starch  or  glycogen  do  not 
hydrolyze  maltose,  so  that  if  maltose-splitting  ferments  be  absent 
the  process  of  hydrolysis  ceases  at  this  stage. 

Maltose  is  highly  dextrorotatory,  exhibits,  mutarotation,  reduces 
Fehling's  solution  and  forms  a  phenylosazone.  When  hydrolyzed  by 
acids  it  yields  two  molecules  of  glucose,  but  it  is  much  less  readily 
hydrolyzed  by  acids  than  cane-sugar.  The  ferment  Diastase,  which 
hydrolyzes  starch  and  glycogen,  the  ferment  Invertase  which  hydrolyzes 
cane-sugar,  the  ferment  Lactase  which  hydrolyzes  milk-sugar,  and  the 
ferment  Emulsion  which  hydrolyzes  amygdalin  and  isomaltose,  are  all 
without  action  upon  maltose,  which  is  hydrolyzed  only  by  a  ferment 
known  as  Maltase,  found  in  many  animal  tissues  and  in  the  majority  of 
yeasts.  Maltose  itself  does  not  undergo  alcoholic  fermentation,  and 
must  first  be  split  by  maltase  or  by  acids  into  glucose,  but  as  the 
majority  of  yeasts  contain  maltose,  these  yeasts  can  accomplish  the 
reduction  of  alcohol  from  maltose. 

The  glucose  which  maltose  yields  upon  hydrolysis  is  initially  highly 
rotatory;  on  adding  ammonia  the  rotation  falls.  Hence  maltose  is  a 
derivative  of  a  glucose.  It  is,  in  fact,  glucose-/*  glucoside.  It  can, 
of  course,  exist  in  two  forms,  according  to  whether  the  glucose  moiety 
which  still  contains  a  potential  aldehyde  group  is  in  the  a  or  )8  form. 
The  one  maltose,  a-maltose,  is  therefore  a-glucose-a-glucoside;  the 
other  is  /3-glucose-a-glucoside. 

Maltose  can  be  synthesized  from  glucose  by  the  condensing  action 
of  strong  acids.  But  in  addition  to  maltose  another  disaccharide  is 
obtained  by  this  process.  This  disaccharide  is  isomeric  with  maltose 
and  yields,  like  maltose,  two  molecules  of  glucose  on  hydrolysis, 
differs  from  maltose  in  the  characteristics  of  its  phenylosazone,  and 
also  in  the  fact  that  it  is  not  fermentable  by  yeasts.  The  ferment 
maltase,  in  fact,  has  no  action  upon  it,  while  the  ferment  emulsion, 

1  Marchlcwski  in  1899. 


DISACCHARIDES  79 

which  is  found  in  certain  plant-tissues  and  which  has  no  action  upon 
maltose,  hydrolyzes  this  sugar  with  the  production  of  .two  molecules 
of  glucose.  This  glucose,  unlike  the  glucose  which  is  produced  in  the 
hydrolysis  of  maltose,  is  of  low  initial  rotatory  power.  On  adding  a 
drop  of  ammonia  to  its  solution  the  rotatory  power  increases.  Hence, 
this  sugar,  which  is  called  Isomaltose,  is  a  derivative  of  0-glucose.  It  is 
a  mixture  of  a-glucose-/3-glucoside,  and  /3-glucose-/3-glucoside. 

Milk-sugar,  also  called  Lactose,  has  not  been  encountered  in  the 
vegetable  kingdom.  It  does  not  occur  preformed  in  any  item  of  our 
diet  excepting  in  milk,  nor  does  it  appear  likely  that  one  of  its  con- 
stituent hexoses,  galactose,  is  commonly  obtainable  from  any  other 
dietary  source  than  milk.  Of  course  it  might  be  obtained  from  brain- 
tissue,  but  this  cannot  be  regarded  as  a  customary  item  of  our  dietary. 
Lactose  yields,  on  hydrolysis,  one  molecule  of  d-glucose  and  one  of 
d-galactose.  Lactose  exhibits  mutarotation,  reduces  Fehling's  solution, 
and  forms  a  phenylosazone.  Lactose  is  not  hydrolyzed  by  maltase, 
invertase,  diastase  or  emulsin,  but  it  is  hydrolyzed  by  a  specific  fer- 
ment designated  Lactase,  and  found  in  the  gastric  mucous  membrane 
and  in  a  few  yeasts  such  as  Kephir  yeast.  This  yeast  is  employed  by 
the  Arabs  to  make  a  sparkling  alcoholic  beverage,  "Kephir,"  from 
the  milk  of  mares. 

Milk-sugar  is  found  in  varying  amounts  in  the  milk  of  all  mammals. 
During  pregnancy  it  is  often  found  in  small  quantities  in  the  urine; 
and  after  weaning  it  also  tends  to  escape  for  a  few  days  through  the 
kidneys.  Extirpation  of  the  mammary  glands  in  milch-goats  and 
cows  gives  rise  to  a  notable  increase  in  the  amount  of  sugar  in  the 
blood  (Glucohemia)  and  also  to  the  appearance  of  glucose  in  the  urine 
(Glycosuria)  .  It  thus  appears  probable  that  in  the  mammary  glands 
milk-sugar  is  formed  from  glucose  alone,  and  not  from  glucose  and 
galactose  in  the  diet.  On  comparing  the  formulae  of  glucose  and 
galactose: 

HCOH  HCOH 

-    ;: 


HCOH\  \                       HCOH 

>  °< 

HOCH  /  \  HOCH 

CH  CH 

HCOH  HCOH 


CH2OH  CH2OH 

d-glucose.  d-galactose. 

it  will  be  seen  that  the  transformation  of  galactose  into  glucose  involves 
the  rupture  of  the  oxide-ring  and  its  closure  again  on  the  opposite  side. 


80    DISACCHARIDES,  POLYSACCHARIDES  AND  GLUCOSIDES 

No  enzyme  has  yet  been  isolated  which  is  capable  of  bringing  about 
this  transformation. 

It  is  for  this  reason  and  possibly  also  for  others  connected  with  the 
metabolism  of  the  intestinal  bacteria,  that  maltose  or  cane-sugar  can- 
not be  regarded  as  satisfactory  substitutes  for  milk-sugar  in  the  diet 
of  young  infants.  On  the  other  hand,  the  assimilation-limit  or  quantity 
which  may  be  ingested  at  once  without  leading  to  alimentary  glycosuria, 
is  lower  for  lactose  than  for  the  other  sugars,  so  that  if  large  quantities 
of  sugar  have  to  be  given  to  make  up  the  requisite  calorific  value  of  the 
diet,  as  in  the  case  of  fat-intolerant  infants,  maltose  may  be  used  as  an 
accessory  to  lactose  in  the  food.  Cane-sugar  being  the  disaccharide 
which  is  most  foreign  to  animal  tissues,  and  also  the  sweetest  in  taste, 
is  much  less  suitable  than  milk-sugar  or  maltose  for  the  dietary  of 
young  infants. 

The  galactose  which  milk-sugar  yields  on  hydrolysis  is  of  low  rotatory 
power,  and  its  rotation  increases  on  adding  ammonia.  Hence  lactose 
is  glucose-/3-galactoside,  since  it  can  be  shown  by  forming  the  osazone 
of  the  sugar  and  hydrolyzing,  when  the  phenylhydrazine  remains 
attached  to  the  sugar  with  the  free  (potential)  aldehyde  group,  that 
it  is  the  glucose  radical  which  contains  the  potential  aldehyde-group, 
the  aldehyde-group  of  the  galactose  offering  the  point  of  union  for  the 
glucose  molecule.  The  potential  aldehyde-group  of  the  glucose  radical 
can  exist  either  in  the  a-  or  the  /3-form.  The  a-lactose  (rotatory  power 
=  -f-  86°)  is  therefore  a-glucose-jft-galactoside,  while  ,8-lactose  (rotatory 
power  =  +  35°)  is  /3-glucose  =  0-galactoside.  No  derivative  of  a- 
galactose  is  certainly  known  to  occur  in  nature.  If,  however,  kephir 
lactase  be  allowed  to  act  upon  a  concentrated  mixture  of  equal  parts  of 
glucose  and  galactose,  two  isomeric  lactoses  are  produced,  both 
exhibiting  mutarotation,  and  both  yielding  d-glucose  and  d-galactose 
on  hydrolysis.  One  of  these  is  ordinary  lactose,  the  other  has  been 
termed  Isolactose  and  is  possibly  a  mixture  of  a-  and  0-glucose-a:- 
galactosides. 

Each  of  the  disaccharides  which  contains  a  potentially  active 
aldehyde-group  can,  therefore,  exist  in  four  different  forms.  Thus  for 
maltose  we  have: 

a     glucose-a-glucoside    \    a- 

0     glucose-a-glucoside    /    ft-     maltose 

a     glucose-/3-glucoside    \    a-     .          ., 
ft     glucose-0-glucoside    /    ft-     ^omaltose 

and  for  lactose  we  have: 

a     glucose-a-galactoside    \    a-     .    , 

ft     glucose-a-galactoside    /    ft-     lsolactose 

a     glucose-/3-galactoside    \    a-     . 
ft     glucose-0-galactoside    /   ft-     lact< 


POL  YSACCHA  RIDES  81 

These  relationships  are  very  important,  and  we  shall  have  occasion 
to  refer  to  them  again  in  later  chapters. 

Melibiose  is  a  galactoside  of  glucose.  It  is  derived  from  the  trisac- 
charide  raffinose  by  hydrolysis. 


POLYSACCHARIDES. 

We  must  now  take  up  the  consideration  of  the  Polysaccharides,  or 
carbohydrates  formed  by  the  union  of  more  than  two  molecules  of  the 
simple  sugars,  with  the  elimination  of  a  corresponding  number  of 
molecules  of  water. 

A  few  tri-  and  tetra-saccharides  are  tolerably  well  known  and 
defined;  of  these  the  most  important  is  Raffinose,  CigH^Oie,  a  trisac- 
charide  which  is  found  abundantly  in  many  plant-tissues  and  products, 
particularly  molasses,  eucalyptus-manna,  wheat,  barley,  fungi,  bacteria 
and  yeast.  It  may  be  distinguished  from  cane-sugar  by  its  greater 
solubility  in  methyl  alcohol,  and  by  the  fact  that  it  is  split  by  emulsin, 
yielding  d-fructose  and  melibiose,  while  cane-sugar  is  not  attacked  by 
this  ferment.  Hydrolysis  by  acids  yields  first  d-fructose  and  melibiose, 
then  the  melibiose  is  hydrolyzed  more  slowly,  yielding  d-galactose  and 
d-glucose.  Raffinose  does  not  reduce  Fehling's  solution. 

Raffinose  is  not  split  by  animal  tissue-extracts  nor  by  any  of  the 
digestive  juices  with  the  exception  of  gastric  juice  which  slowly  inverts 
it  owing  simply  to  the  fact  of  its  acidity  and  not  to  any  ferment  con- 
tained in  the  juice.  As  the  gastric  contents  are  only  distinctly  acid  for 
a  brief  period  during  digestion  we  may  infer  that  this  mode  of  splitting 
raffinose  is  of  no  nutritive  significance  since  it  must  be  of  very  trivial 
extent.  A  portion  of  the  raffinose  contained  in  the  food  is  probably 
absorbed  unaltered  and  excreted  as  such  in  the  urine,  the  remainder 
with  the  exception  of  the  very  small  proportion  inverted  in  the  stomach, 
remains  unaltered  until  it  reaches  the  large  intestine  (cecum)  where  it 
is  rapidly  inverted  by  the  bacteria  which  inhabit  this  portion  of  the 
alimentary  canal  and  is  thus  rendered  available  for  nutritive  purposes. 

We  here  meet  with  a  phenomenon  which  is  yearly  growing  of  greater 
significance  in  our  eyes,  namely  the  Symbiotic  Relationship  between 
the  mammals  and  the  bacterial  parasites  which  inhabit  their  intestines. 
While  the  bacterial  flora  of  the  intestines  constitute  a  parasitic  growth, 
yet  their  tenure  of  the  intestine  is  not  wholly  to  the  disadvantage  of  the 
host,  and  through  the  multifarious  enzymes  which  they  produce  these 
organisms  render  available  to  mammals  foodstuffs  which  would  other- 
wise be  indigestible  and  excreted  unaltered.  It  is  probably  for  this 
reason  that  chickens  and  rats  fed  upon  a  strictly  aseptic  diet  do  not 
grow  normally.  While  in  the  instance  chosen,  that  of  raffinose,  the 
products  thus  rendered  available  may  not  be  of  indispensible  impor- 
tance to  the  animal  economy,  yet  in  many  cases,  as  for  example  in  the 
splitting  of  chlorophyll  by  the  intestinal  bacteria,  the  products  which 
6 


82     DISACCHARIDES,  POLYSACCHARIDES  AND   GLUCOSIDES 

result  (containing  methyl-pyrrole  groupings),  may  very  possibly  be 
unobtainable  by  mammals  in  any  other  way. 

The  higher  polysaccharides  are  very  imperfectly  defined.  We  have 
no  reliable  methods  which  are  available  for  determining  their  molec- 
ular weights,  and  we  do  not  know,  therefore,  how  many  molecules  of 
sugar  take  part  in  their  formation.  The  group  is  a  very  large  one,  and 
the  general  formula  (C6H10O5)n  may  be  ascribed  to  the  majority  of  its 
best-known  members,  indicating  that  they  are  formed  by  the  union 
of  an  indefinite  number,  n,  of  hexose  anhydrides.  The  following  sub- 
stances are  important  and  typical  members  of  the  group:  Starch, 
Glycogen,  Dextrins,  Inulin,  Pectin,  Humin,  Cellulose,  Gums,  and  Vege- 
table Mucilages.  It  is  very  important  to  recollect,  however,  that  these 
are  merely  arbitrary  terms  used  to  describe  very  ill-defined  members 
of  the  series.  Thus  we  cannot  be  certain  that  there  is  only  one  chemical 
individual  Starch ;  on  the  contrary,  it  appears  probable  that  there  may 
be  many  starches,  and  starch  is  certainly  known  in  two  widely  different 
forms,  to  wit :  a  form  insoluble  in  water  and  a  form  which  is  soluble  in 
water.  On  the  other  hand  it  should  be  recollected  that  the  differences 
which  are  observed  between  these  forms  of  starch  may  possibly  be 
purely  physical,  and  not  chemical  differences  at  all.  We  here  encounter, 
in  fact,  a  problem  which  is  presented  generally  by  the  colloids,  and 
which  we  shall  meet  with  again  in  connection  with  the  proteins. 

Starch,  inulin,  gums,  mucilages  and  glycogen  do  not  reduce  metallic 
oxides  in  alkaline  solutions.  They  do  not,  therefore,  contain  potentially 
active  aldehyde-groups.  Dextrins,  on  the  contrary,  do  contain  alde- 
hyde-groups, for  they  reduce  Fehling's  solution.  With  the  possible 
exceptions  of  glycogen  and  inulin,  the  polysaccharides  do  not  form 
crystals,  or  at  least,  they  have  not  as  yet  been  prepared  in  crystalline 
form.  Water  dissolves  some  of  them,  others  only  swell  in  cold  water 
and  dissolve  in  hot  water,  others  are  unaffected  by  water.  Solutions 
of  the  polysaccharides  do  not  taste  sweet  unless  held  in  the  mouth  for 
a  sufficient  period  to  enable  the  diastase  (Ptyalin)  in  the  saliva  to 
bring  about  hydrolysis.  Solutions  of  the  polysaccharides  are  optically 
active.  The  higher  polysaccharides  do  not  diffuse  through  parchment 
paper,  thus  behaving  typically  as  colloids.  They  do  not  form  com- 
pounds with  phenylhydrazine. 

The  polysaccharides  play  a  wide  variety  of  parts  in  the  vegetable 
kingdom.  In  the  first  place,  they  serve  as  reserve  materials,  or  stores 
of  sugar,  laid  up  against  a  future  time  of  need;  such  a  part  is  that 
played  by  starch  (or  vegetable  glycogen,  as  it  may  be  called  in  analogy 
to  animal  glycogen,  which  plays  a  similar  part  in  the  animal  economy) 
and  also  by  inulin.  The  gums  and  mucilages,  on  the  contrary,  serve, 
in  part  at  least,  to  close  up  injuries  and  protect  them  while  healing. 
The  celluloses,  again,  have  yet  another  function  to  perform.  They, 
or  their  derivatives,  constitute  the  supporting  tissues  of  plants,  just  as 
bones  or  exoskeletons  constitute  the  supporting  tissues  of  animals. 

The  Celluloses  (the  plural  is  employed  because  it  appears  highly 


POLYSACCHARIDES  83 

probable  that  there  are  many  modifications  of  cellulose),  are  insoluble 
in  all  ordinary  solvents,  such  as  water,  alcohol,  ether,  and  so  forth. 
Cellulose  dissolves,  however,  in  solutions  of  many  metallic  salts  in  the 
presence  of  excess  of  strong  acid,  for  example  in  zinc  chloride  in  acid 
solution,  and  in  the  hydrochloric  acid  solutions  of  antimony,  mercuric, 
or  bismuth  chlorides.  The  requisite  condition  for  solution  appears  to 
be,  the  presence  of  a  salt  of  a  weak  metallic  base  in  acid  solution. 
Another  solvent  for  cellulose  is  an  ammoniacal  solution  of  cupric 
oxide,  known  as  "  Schweitzers'  Reagent."  In  the  presence  of  con- 
centrated sulphuric  acid,  sulphuric-acid  esters  of  cellulose  are  formed 
and  pass  into  solution.  If  this  solution  be  diluted  and  boiled,  glucose 
is  formed  and  glucose  only,  hence  cellulose  is  an  anhydride  of  glucose. 
A  preliminary  stage  in  this  hydrolysis  is  the  formation  of  Amyloid, 
a  soluble  colloidal  substance  which  resembles  starch  in  yielding  a  blue 
color  with  iodine. 

Cellulose  is  indigestible  by  any  of  the  ferments  contained  in  or 
produced  by  mammalian  tissues.  It  is,  however,  digestible  by  bacteria, 
and  as  much  as  seventy  per  cent,  of  unlignified  cellulose  may  be  dis- 
solved in  vitro  by  the  juices  from  the  lower  intestine  of  the  horse.  The 
products  of  this  form  of  digestion  are  not  sugars,  but  carbon  dioxide, 
methane  and  fatty  acids.  Human  beings  have  been  found  to  utilize 
as  much  as  forty  per  cent,  of  young  and  tender  cellulose,  doubtless 
through  the  agency  of  the  intestinal  bacteria.  Hence  the  nutritive 
value  of  cellulose,  especially  in  animals  such  as  the  cow  and  horse 
which  possess  very  long  intestines,  is  by  no  means  negligible.  But 
the  celluloses  are  of  significance  to  the  animal  economy  from  yet 
another  point  of  view.  By  virtue  of  their  incomplete  digestibility 
they  communicate  bulk  and  substance  to  the  f eces  and  thus  facilitate 
their  passage  through  the  intestines,  in  the  first  place  by  bringing  about 
a  favorable  distention  of  the  intestinal  muscular  walls,  and  in  the 
second  place  by  furnishing  these  muscles  with  material  upon  which  to 
exert  leverage.  Prior  to  the  introduction  of  "War-breads"  the  ten- 
dency of  our  times  was  to  eliminate  indigestible  carbohydrates  more  and 
more  thoroughly  from  the  diet  and  the  prevalence  of  intestinal  stasis 
and  chronic  constipation  in  modern  communities  is  doubtless  attri- 
butable, in  part  at  least,  to  this  "refinement"  of  our  foodstuffs.  A 
crude  endeavor  to  correct  this  deficiency  in  our  diet  is  frequently 
made  by  mixing  bran  or  other  coarsely  ground  cellulose-rich  materials 
with  the  flour  from  which  bread  is  made.  This  remedy  may  in  many 
instances,  however,  be  worse  than  the  disease,  for  the  ingestion  of  large, 
horny  and  sharp-edged  indigestible  fragments  with  the  food  may  lead 
to  lacerations  of  the  intestine,  and  consequent  inflammatory  reactions 
or  enteritis.  What  is  required  is  finely  ground  cellulose-rich  material, 
such  as  our  ancestors  enjoyed  when  they  ground  up  their  grains  by 
hand  between  two  hard  stones.  Agar  is  frequently  employed  to  com- 
municate indigestible  bulk  to  the  diet  or,  in  recent  years,  heavy  taste- 
less petroleum  oils,  but  in  administering  these  substances  we  are  merely 


84    DISACCHARIDES,  POLYSACCHARIDES  AND  GLUCOSIDES 

striving  to  remedy  the  consequences  of  a  totally  unnecessary  dietary 
habit  which  arises  from  a  threefold  origin  of  public  ignorance,  a 
fancied  superiority  of  things  which  are  white,  and  therefore  "pure," 
white  bread,  white  eggs,  white  (i.  e.,  sulphured)  dried  fruits,  white 
sugar  (made  to  appear  white  by  the  addition  of  litmus)  and  so  forth, 
and  in  the  irresponsible  self-interest  of  millers  and  bakers.  The  War, 
through  the  introduction  of  more  thorough  utilization  of  grains  to  make 
"War  flour"  and  "War  bread"  has  thus  no  doubt  proved  a  veritable 
blessing  in  disguise  to  many  chronic  sufferers  and  it  is  not  improbable 
that  the  reinstatement  of  our  former  foolish  and  wasteful  habits  of 
milling  will  be  prevented  or  delayed  by  a  more  general  public  recog- 
nition of  the  beneficial  role  of  indigestible  residues  in  the  food. 

Cellulose  occurs  almost  exclusively  in  the  vegetable  kingdom.  It 
is  found,  however,  in  the  shells  of  Tunicata.  Otherwise  it  is  unknown 
in  the  animal  kingdom.  In  the  cell- walls  of  plants,  not  only  true  cellu- 
lose is  found  but  other  cellulose-like  substances,  some  of  which  yield 
not  only  glucose,  but  other  sugars,  even  pentoses  such  as  arabinose, 
xylose,  etc.  Polysaccharides  which  yield  only  pentoses  on  hydrolysis 
are  also  found,  and  are  known  as  Pentosans. 

As  the  cell-walls  of  plants  advance  in  age  they  undergo  a  peculiar 
change  resulting  in  the  acquirement  of  greater  rigidity.  This  process 
is  known  as  Lignification.  The  exact  nature  of  the  change  which  occurs 
is  not  known,  but  it  has  been  suggested  that  Lignin  is  formed  from 
cellulose  by  the  formation  of  compounds  with  aromatic  derivatives. 

The  vegetable  gums  and  mucilages  are  a  very  heterogeneous  group 
of  polysaccharides.  The  gums  are  insoluble,  the  mucilages  soluble  in 
water.  The  majority  of  them  yield  galactose  and  arabinose  when  hydro- 
lyzed  by  dilute  acids.  Agar-agar,  so  widely  used  in  culture-media  for 
bacteria,  is  a  representative  of  this  class  of  carbohydrates;  it  is  derived 
from  certain  marine  algae.  From  marine  algae  of  the  Fucus  type  is 
also  obtained  a  polysaccharide  yielding  pentoses  on  hydrolysis  which  is 
designated  Algin.  It  is  a  colloidal  substance  which  behaves  like  a  weak 
acid,  forms  insoluble  salts  of  aluminium  and  lime,  and  is  employed  as 
a  waterproofing  material  and  a  substitute  for  size. 

Closely  related  to  the  gums  and  mucilages  is  a  group  of  substances, 
the  Pectins,  which  are  of  very  great  industrial  importance  inasmuch 
as  they  are  responsible  for  the  gelation  of  fruit-jellies.  The  pectins 
are  white  amorphous  gelatinous  substances,  which  form  colloidal  solu- 
tions in  water,  do  not  reduce  Fehling's  solution,  and  yield  galactose, 
glucose  and  pentoses  on  hydrolysis  by  acids.  They  are  believed  to  be 
derived  by  partial  hydrolysis,  due  to  the  organic  acids  present  in 
fruit-extracts,  from  a  series  of  parent-substances,  the  Pectoses,  which 
are  present  in  plant-tissues  in  the  form  of  insoluble  calcium  salts. 
The  pectins  are  converted  by  dilute  alkalies  or  by  the  ferment  Pectase 
into  Pectic  Acid,  the  calcium  salt  of  which  is  insoluble  in  water  and 
forms  jellies.  Since  pectase  is  destroyed  by  heat,  the  formation  of 
fruit-jellies  by  extracting  fruits  with  hot  sugar-solutions  is  not  to  be 


POLYSACCHARIDES  85 

attributed  to  the  action  of  pectase,  but  rather  to  the  production  of 
insoluble  jelly-forming  substances  from  pectose  or  pectin  by  the 
hydrolyzing  action  of  the  fruit-acids.  The  pectins  are  not  hydrolyzed 
by  diastases,  they  are,  however,  hydrolyzed  by  special  enzymes,  the 
Pectinases,  found  in  malt  and  in  certain  moulds  which  liquify  pectin 
jellies  with  the  production  of  reducing  sugars. 

Coming  now  to  those  polysaccharides  which  are  primarily  of  nutri- 
tive importance,  Starch  is  the  form  in  which  sugar  is  chiefly  stored  up 
by  plants  for  future  consumption,  although  cane-sugar,  inulin  and 
other  carbohydrates  frequently  play  a  similar  part. 

Starch  is  found  in  the  greatest  amounts  in  those  portions  of  plants 
which  are  subsequently  to  be  drawn  upon  for  the  materials  of  growth. 
Thus  seeds,  roots,  bulbs,  tubers  and  the  pith  of  deciduous  trees  in 
winter  are  particularly  rich  in  starch,  this  carbohydrate  frequently 
comprising  as  much  as  eighty  per  cent,  of  the  dry  weight  of  the  material. 
The  starch  is  stored  up  in  these  tissues  in  the  form  of  stratified  granules, 
which  differ  characteristically  in  form  and  size  in  different  plants.  It 
is  by  means  of  these  characteristics  of  form,  size  and  stratification  of  the 
granules  that  we  can  tell  very  readily  whether  a  starch  alleged  to  have 
been  derived  from  one  specified  source  has  or  has  not  been  adulterated 
in  the  pursuance  of  "legitimate  business  enterprise"  with  starch 
derived  from  some  other  and  cheaper  source. 

The  concentric  rings,  or  stratifications  of  starch-grains  represent 
their  gradual  growth,  and  intimate  that  the  growth  of  starch-grains 
takes  place  rhythmically,  periods  of  desposition  alternating  with 
periods  of  rest.  Starch  is  only  slightly  and  very  slowly  changed  by 
cold  water,  but  in  hot  water  the  grains  swell  up  and  finally  burst, 
forming  what  is  known  as  "starch-paste."  Neither  starch  nor  starch- 
paste  reduces  metallic  oxides  in  alkaline  solution. 

A  very  familiar  test  for  starch  is  the  formation  of  a  very  deep  indigo- 
blue  coloration  when  it  is  acted  upon  by  iodine  solutions  in  the  presence 
of  hydriodic  acid  or  of  an  iodide.  The  color  disappears  on  boiling  and 
reappears  on  cooling.  In  applying  this  test  it  is  necessary  to  remember 
that  it  is  not  given  in  the  presence  of  excess  of  reagents  which  are 
oxidized  by  iodine,  such  reagents,  for  example,  as  hydroxides  of  the 
alkalies,  or  sulphurous  or  arsenous  acids.  It  is  in  connection  with 
this  test  that  we  meet  with  very  clear  indications  that  starch  is  not  a 
homogeneous  chemical  unit,  for  varieties  of  starch  are  known  which 
do  not  give  a  blue,  but  a  reddish-brown  or  a  "port-wine"  color  with 
iodine.  We  do  not  know  to  what  these  colorations  are  due,  or  whether 
they  are  specific,  i.  e.,  yielded  by  one  chemical  individual  alone,  or 
generic,  i.  e.,  yielded  by  a  group  of  similar  chemical  individuals. 

On  boiling  starch  with  dilute  mineral  acids,  glucose  and  only  glucose 
is  obtained.  Starch  is  therefore  an  anhydride  of  glucose.  If  the  acid  is 
allowed  only  to  act  upon  the  starch  in  the  cold,  or  with  very  gentle 
heating,  a  modification  of  starch,  known  as  "soluble  starch"  is  obtained. 
If  we  act  upon  starch  for  several  weeks  with  cold  dilute  mineral  acids, 


86     DISACCHARIDES,  POLYSACCHARIDES  AND  GLUCOSIDES 

or  for  an  hour  with  four  per  cent,  sulphuric  acid  at  80°  (/.,  we  obtain 
"Amylodextrin,"  which  yields  a  port-wine  coloration  with  iodine. 
Further  hydrolysis  of  the  amylodextrin  yields  a  mixture  of  simple 
dextrins  which  give  no  color  with  iodine  ("  Achroodextrin") ;  still  further 
hydrolysis  yields  Glucose,  an  intermediate  product  of  hydrolysis  being 
Maltose  which,  however,  in  the  acid-hydrolysis  of  starch,  is  immediately 
broken  down  into  glucose,  so  that  in  the  hydrolysis  of  starch  by  acids 
maltose  is  only  transiently  present  in  the  system.  In  the  hydrolysis 
of  starch  by  diastatic  ferments,  however,  unless  Maltase  be  also  present, 
the  final  product  of  hydrolysis  is  the  disaccharide  maltose,  the  inter- 
mediate stage  of  hydrolysis  being  so  far  as  we  know,  similar  to  those 
observed  in  the  hydrolysis  of  starch  by  acids.  The  hydrolysis  of 
starch  takes  place,  therefore,  step  by  step,  with  the  production  of 
intermediate  stages  of  hydrolysis  before  the  final  product,  glucose, 
is  obtained.  We  shall  meet  with  analogous  phenomena  among  the 
proteins,  and  if  we  draw  a  parallel,  which  is  of  course  only  justifiable  in 
a  formal,  not  in  a  chemical  sense,  between  the  hydrolysis  of  starch  and 
the  hydrolysis  of  proteins,  then  we  would  have  the  following  table  of 
analogues: 

Starch  analogous  to         Proteins 


"Soluble  starch' 

Amylodextrin 

Achroodextrins 

Maltose 

Glucose 


Albumoses 

Peptones 

Polypeptids 

Dipeptids 

Amino-acids 


Inulin,  a  polysaccharide  found  in  the  tubers  of  dahlias,  and  in  other 
situations,  bears  the  same  relationship  to  fructose  that  starch  does  to 
glucose.  On  hydrolysis  by  acids  it  yields  only  fructose;  it  is  not 
hydrolyzed  by  any  of  the  diastatic  ferments  which  hydrolyze  starch 
or  glycogen. — It  is>  however,  hydrolyzed  by  a  special  ferment  Inulinase. 
Inulin  differs  very  markedly  from  starch,  in  that  it  dissolves  readily 
in  warm  water  with  the  formation  of  a  solution  instead  of  a  paste,  and 
it  yields  a  yellow  color  with  iodine. 

In  various  other  situations  in  the  vegetable  kingdom  other  poly- 
saccharides  resembling  starch  and  inulin  are  found,  differing  from  these, 
however,  in  certain  characteristics.  Thus  we  have  Amylin,  Lavosin, 
Cerosin,  and  Secalin,  etc.,  found  in  grain-seeds,  some  of  which  yield 
glucose  on  hydrolysis,  others  fructose.  In  Lupinus  luteus  is  found 
Galactin,  a  polysaccharide  which  yields  only  galactose  on  hydrolysis. 
In  Lichens  is  found  a  polysaccharide,  Lichenin,  which  yields  only 
glucose  on  hydrolysis  by  acid,  but  which,  curiously  enough,  is  not 
hydrolyzed  by  diastatic  ferments.  It  yields  a  yellow  color  on  treat- 
ment with  iodine. 

Glycogen  is  to  the  animal  economy  what  starch  is  to  that  of  the  plant. 
It  was  observed  by  the  distinguished  French  investigator,  Claude 
Bernard,  in  1848,  that  the  sugar-content  of  the  liver,  excepting  after 
starvation,  is  very  high.  He  further  found  that  the  sugar  which  the 


POLYSACCHARIDES  87 

liver  yields  on  standing  is  not  present  as  such,  but  in  a  form  resembling 
starch,  which  is  rapidly  hydrolyzed  by  enzymes  contained  in  the  tissues, 
or  by  acids,  yielding  glucose.  Glycogen  may  be  prepared  from  fresh 
liver  by  extracting  the  tissues  with  strong  potassium  hydroxide  solu- 
tion, which  decomposes  the  proteins  but  does  not  hydrolyze  the 
glycogen,  and  then  precipitating  with  alcohol.  If  the  liver  be  allowed 
to  stand  before  extraction,  a  much  smaller  quantity  of  glycogen  will 
be  obtained,  and  simultaneously  it  will  be  found  that  sugar  has 
appeared  in  the  liver.  If  the  liver  be  heated  to  boiling  before  being 
allowed  to  stand,  the  glycogen  does  not  disappear  and  no  increase 
in  the  sugar  content  of  the  liver  is  observed.  Evidently,  therefore 
the  disappearance  of  glycogen  in  the  liver  on  standing  is  due  to  the 
action  of  a  hydrolyzing  ferment  which  is  destroyed  or  inactivated  by 
heating. 

Glycogen,  although  like  starch,  an  anhydride  of  glucose  is  never- 
theless readily  and  sharply  distinguishable  from  starch.  It  forms  when 
pure  a  fine  white  amorphous  powder.  Its  molecular  weight  is  unknown. 
It  dissolves  in  cold  water,  forming  opalescent  solutions,  but  it  is  a 
typical  colloid  and  does  not  diffuse  through  parchment.  With  iodine 
glycogen  yields  a  reddish-brown  or  port-wine  coloration  which  dis- 
appears on  heating  and  reappears  on  cooling. 

The  hydrolysis  of  glycogen,  like  that  of  starch,  takes  place  in  step- 
like  stages.  Intermediate  products  of  hydrolysis  are  dextrins  and 
maltose.  In  the  absence  of  maltase  the  diastatic  ferments  hydrolyze 
it  as  far  as  the  maltose-stage  and  then  their  action  stops.  It  is  not  by 
any  means  certain  that  there  is  only  one  glycogen  or  that  there  are  not 
a  variety  of  different  reserve-carbohydrates  in  animal  tissues,  but  if 
this  is  the  case  no  means  has  yet  been  found  of  positively  separating 
and  identifying  them. 

Glycogen  is  found  in  a  variety  of  tissues,  but  the  chief  storehouses 
in  the  vertebrates  are  the  liver  and  the  muscles.  In  invertebrata 
glycogen  occurs  in  organs  which  correspond  in  function  to  the  liver. 
It  also  occurs  in  the  protoplasm  of  unicellular  animals  and  is  abundant 
in  yeast.  It  appears  never  to  occur  in  the  nucleus. 

The  glycogen  which  is  stored  up  in  the  striated  and  smooth  muscles 
of  the  vertebrata  is  of  peculiar  significance,  in  that  it  stands  quantita- 
tively in  direct  relation  to  the  work  which  the  muscles  perform.  As 
the  muscles  do  work,  glycogen  disappears  from  them.  As  might  be 
expected,  therefore,  the  percentage  of  glycogen  in  muscle  varies  very 
much  in  different  animals  and  under  different  conditions.  The  follow- 
ing figures,  given  by  Cramer,  show  this  very  clearly: 

Glycogen, 
Animal.  Muscle.  per  cent. 


Dog  Number  1 
Dog  Number  2 
Dog  Number  3 
Dog  Number  4 


Biceps  brachii  0.17 

Quadriceps  femoris  0.53 

Biceps  brachii  0.25 

Quadriceps  femoris  0.32 

Dorsal  muscul  ature  0.135 

Posterior  adductors  0 . 077 

Dorsal  musculature  0.417 

Posterior  adductors  0 . 444 


88     DISACCHARIDES,  POLYSACCHARIDE8  AND  GLUCOSIDES 

Glycogen  is  also  found  in  glandular,  epithelial  and  connective  tissues 
and  in  the  brain.  The  distribution  of  glycogen  in  the  body  is  very 
variable;  the  following  figures  were  obtained  by  Schondorff,  employing 
dogs  which  had  been  well  fed  with  carbohydrates  and  meat  shortly 
before  death : 

One  hundred  grammes  of  glycogen  were  distributed  in  different 
parts  of  the  body  in  the  following  proportion  in  seven  dogs  employed : 

Minimum  Minimum 

observed.  observed.  Average. 

Blood 0.04  0.001  0.015 

Liver 56.74  20.09  37.97 

Muscle 62.55  31.22  44.23 

Bone .  12.88  5.36  9.25 

Skin 11.38  1.42  4.49 

Viscera 7.30  0.38  3.81 

Heart 0.28  0.08  0.17 

Brain 0.23  0.04  0.09 

It  will  be  observed  that  the  heart-muscle,  which  is  in  continual 
activity,  contains  very  little  reserve-stock  of  carbohydrates.  It  is 
evidently  unable  to  accumulate  a  reserve  or  capital  of  carbohydrate 
and  maintains  its  activity  upon  its  current  income.  With  this  may  be 
correlated  the  fact  that  after  each  beat  of  the  heart  a  definite  and 
relatively  lengthy  period  occurs,  the  "refractory  period"  during  which 
even  the  application  of  stimuli  fails  to  elicit  a  contraction  from  the 
heart-muscle,  whereas  ordinary  striated  muscle,  containing  abundant 
stores  of  reserve  carbohydrate,  may  be  stimulated  repeatedly  at  exceed- 
ingly brief  intervals  until  relaxation  between  the  contractions  becomes 
a  mechanical  impossibility,  and  the  contractions  fuse  into  one  "tetanic" 
contraction  which  relaxes  only  when  the  muscle  becomes  exhausted 
and  its  stores  of  glycogen  depleted. 

It  will  be  noted,  also,  that  the  percentage  of  glycogen  in  the  blood 
is  extraordinarily  low.  In  fact  it  appears  that  the  only  form  in  which 
carbohydrate  material  circulates  in  the  Vertebrata  is  that  of  glucose, 
and  that  this  is  also  the  only  form  in  which  carbohydrate  food  is 
utilized  by  the  tissues  for  the  production  of  energy,  or  the  manufacture 
of  reserve-materials.  Now  the  carbohydrates  of  the  food  are  usually 
ingested  in  the  form  of  starch,  glycogen,  and  other  polysaccharides,  or 
in  the  form  of  disaccharides,  such  as  cane-sugar  or  lactose,  and  these 
carbohydrates  are  readily  utilized  by  the  organism.  Preparatory  to 
utilization  therefore,  these  carbohydrates  must  undergo  elaborations 
and  transformations  resulting  in  the  formation  of  glucose. 


AMINO  -POLYSACCHARIDES. 

The  hydrolysis  of  proteins  which  contain  a  glucosamin  radical  yields 
in  some  instances  an  amino-disaccharide,  presumably  diglucosamin. 
The  most  important  amino-polysaccharide  in  the  animal  economy  is, 
however,  Chitin,  which  forms  the  exoskeleton  of  the  Jnsecta  and  the 


GLUCOSIDES  89 

Crustacea.  It  may  be  obtained  in  colorless  semi-transparent  lamellae 
which  are  stained  reddish-brown  by  iodine;  on  addition  of  sulphuric 
acid  or  zinc  chloride  the  color  changes  to  blue  or  violet.  Hydrolysis 
with  strong  acids  yields  about  seventy-five  per  cent,  of  d-glucosamin. 
Chitin  also  contains  acetyl  radicals  which  are  liberated  as  acetic  acid 
on  fusion  with  alkali. 

Prolonged  treatment  with  alkali  in  the  cold  leads  to  the  formation 
of  "  soluble  chitin"  which  is  diffusible  through  parchment,  but  has  an 
extraordinary  affinity  for  water,  carrying  the  water  in  the  dialyzer 
with  it  as  it  traverses  the  parchment,  and  withholding  it  from  the 
cavity  of  the  dialyzer  against  hydrostatic  pressure.  Other  products 
of  the  partial  hydrolysis  of  chitin  are  crystallizable  (chitosans). 


GLUCOSIDES. 

The  glucosides  are  a  large  and  important  class  of  substances,  occur- 
ring in  great  variety  in  certain  vegetable  tissues,  and  also  in  exceedingly 
important  tissues  and  localities  in  the  animal  body.  They  yield 
monosaccharides  on  hydrolysis  and  other  radicals  which  differ  widely 
in  different  glucosides. 

Reference  has  already  been  made  to  the  glucoside  Amygdalin  which 
occurs  in  the  kernels  of  cherries  and  almonds  and  is  hydrolyzed  by  the 
ferment  Emulsin,  yielding  glucose,  hydrocyanic  acid  and  benzaldehyde. 
Various  species  of  the  Cruciferce  contain  irritant  glucosides,  notable 
among  which  are  Sinigrin  or  Potassium  Myronate  in  the  oil  of  black 
mustard,  obtained  from  the  seeds  of  Sinapis  nigra,  and  Sinalbin,  in 
the  oil  of  white  mustard,  obtained  from  the  seeds  of  Sinapis  alba. 
Both  sinigrin  and  sinalbin  are  hydrolyzed  in  the  presence  of  water  by  a 
ferment,  Myrosin,  which  occurs  in  the  tissues  of  the  plants  from  which 
they  are  obtained.  The  products  yielded  by  the  two  glucosides  are, 
however,  very  different,  sinigrin  yielding  dextrose,  potassium  bisul- 
phate,  and  allyl  isosulphocyanate,  while  sinalbin  yields  dextrose,  sinapin 
sulphate  (a  sulphate  of  an  alkaloid)  and  methyl  phenyl  isosulpho- 
cyanate. Both  glucosides  are  intensely  irritant  when  applied  to  the 
skin,  and  are  utilized  for  this  purpose  in  therapeutics. 

Glucosides  of  great  therapeutic  importance  are  also  found  in  the 
leaves  and  seeds  of  Digitalis  purpurea,  Strophanthus  and  Scilla,  and 
comprise  the  most  important  active  constituents  of  the  pharmacopceial 
preparations  made  from  these  plants.  They  have  a  characteristic 
action  upon  heart-muscle  of  which  advantage  is  taken  in  the  medical 
treatment  of  cardiac  affections.  The  same  plants  also  contain  gluco- 
sides which  are  either  without  effect  upon  the  heart,  or  else  have  an 
effect  which  is  of  secondary  importance.  Some  of  these  glucosides  are 
members  of  the  saponin  series  and  contribute  to  the  effectiveness  of 
aqueous  extracts  of  the  plants  by  holding  in  solution  substances  which 
would  otherwise  be  insoluble  in  water. 


90    DISACCHARIDES,  POLYSACCHARIDES  AND  GLUCOSIDES 

From  a  biochemical  point  of  view,  and  in  our  present  state  of  knowl- 
edge, perhaps  the  most  noteworthy  glucosides  which  occur  in  plant- 
tissues  are  the  various  members  of  the  Saponin  and  Sapotoxin  group  of 
glucosides  These  substances  are  found  in  a  very  great  variety  of  plant- 
tissues,  but  especially  in  Quillaja,  (soapbark),  Saponaria  (soapwort), 
Cyclamen  (cyclamin),  Solatium  (nightshade  and  potato)  and  Smilax 
(sarsaparilla).  These  glucosides  behave  like  weak  acids  and  are  split 
on  hydrolysis  with  acids  into  sugars  and  other  substances  which  are  for 
the  most  part,  as  yet  undefined.  They  possess  to  a  very  remarkable 
degree  the  property  of  reducing  the  surface-tension  at  surfaces  in  con- 
tact with  water  in  which  they  are  dissolved  and  coating  these  surfaces 
with  an  insoluble  film,  with  the  result  that  the  forces  tending  to  cause 
coalescence  of  bubbles  are  very  much  reduced,  so  that  the  water  con- 
taining saponins  form  froths  like  soap-solutions,  when  it  is  shaken  up 
with  air.  Hence  the  names  "soapbark,"  " soapwort,"  etc.  For  the 
same  reason  they  have  the  property  of  holding  otherwise  insoluble 
substances  in  solution  or  suspension,  since  the  suspended  particles 
have  less  tendency  than  usual  to  clump  together  and  thus  form  masses 
large  enough  to  fall  out  of  the  solution. 

The  saponins  and  solanins  readily  dissolve  or  form  colloidal  solutions 
of  a  variety  of  fatty  substances,  particularly  the  Lecithins,  an  important 
group  of  phosphorus-containing  fatty  substances  which  will  fall  under 
discussion  repeatedly  in  future  chapters.  They  also  form,  in  many 
cases,  insoluble  compounds  with  Cholesterol,  an  aromatic  alcohol,  which 
is  found  associated  with  lecithins  in  all  living  tissues. 

The  power  of  the  saponins  to  dissolve  fatty  substances  is  undoubtedly 
the  origin  of  their  remarkable  action  upon  red-blood  corpuscles,  the 
stroma  of  erythrocytes  being  very  rich  in  lecithins  and  other  fatty 
substances.  As  little  as  one  part  of  cyclamin  added  to  100,000  parts 
of  blood  causes  complete  liquefaction  or  Hemolysis  of  the  stroma  of 
the  corpuscles  with  resultant  setting  free  of  the  enclosed  hemoglobin, 
while  liquefaction  of  a  proportion  of  the  corpuscles  is  brought  about  by 
even  smaller  amounts.  Cholesterol  tends  to  prevent  this  action  of  the 
saponins  by  combining  with  them  to  form  insoluble  compounds,  and 
hence  blood  serum  or  plasma,  since  it  contains  a  small  amount  of 
cholesterol,  to  some  extent  inhibits  the  hemolytic  action  of  the  saponins. 

A  saponin,  digitonin,  which  occurs  in  Digitalis  but  is  devoid  of 
action  upon  the  heart,  is  employed  in  the  quantitative  estimation  of 
cholesterol. 

In  animal  tissues  glucosides  are  found  especially  among  the  decom- 
position-products of  Nucleic  Acids  and  in  the  tissues  of  the  brain. 
The  nucleic  acids  will  fall  under  special  consideration  in  a  later  chapter 
and  it  need  merely  be  stated  here,  in  passing,  that  they  are  phosphoric 
acid  compounds  of  glucosides,  the  Nucleosides,  which  yield  either 
d-glucose  or  d-ribose  and  nitrogenous  bases  on  hydrolysis.  A  nucleo- 
side  is  also  found  in  minute  traces  in  the  blood  and  exerts  an  action 
upon  the  egg-cells  of  the  Sea-urchin  (Strongylocentrotus)  similar  to  that 


CARBOHYDRATE  ESTERS  91 

of  a  saponin;  it  is,  however,  devoid  of  lytic  action  upon  the  red  blood- 
cells  themselves. 

The  glucosides  in  the  brain,  the  Cerebrosides,  occur  in  complex  fatty 
compounds  which  yield  the  free  glucosides,  Phrenosin  and  Kerasin  oil 
partial  hydrolysis.  They  also  exist  in  part  preformed  in  brain-tissue, 
or  at  any  rate  can  be  directly  extracted  therefrom  by  solvents  such 
as  pyridine  or  hot  alcohol  containing  benzole  or  chloroform.  These 
substances  are  not  confined  to  nervous  tissue  but  are  also  present  in 
small  amounts  in  the  kidney  and  liver  and  probably  in  other  organs  as 
well.  They  also  occur  in  the  yolks  of  eggs. 

The  cerebrosides  are  nitrogen-containing  substances  which  are 
hydrolyzed  by  acids,  yielding  fatty  acids,  galactose,  and  a  nitrogenous 
base,  Sphingosine,  which  is  a  diatomic  amino-alcohol  containing  unsatu- 
rated  linkages: 

:  CH.CHOH.CHOH.CH2NH2 


The  fatty  acid  which  is  yielded  by  Phrenosin  is  a  hydroxy-acid, 
Cerebronic  Acid,  C25H5oO3,  while  Kerasin  yields  Lignoceric  Acid,  C^JH^sC^. 
The  two  cerebrosides  differ  furthermore  in  their  solubilities,  phrenosin 
being  almost  insoluble  in  boiling  acetone,  while  kerasin  is  readily 
soluble.  Both  cerebrosides  are  insoluble  in  water  or  in  ether,  but  they 
dissolve  in  hot  alcohol,  from  which  they  crystallize  in  needles  or  plates 
on  cooling.  Solutions  of  phrenosin  are  dextrorotatory,  those  of  kerasin 
being  levorotatory. 

With  sulphuric  acids  the  cerebrosides  yield,  first  a  yellow,  and  later 
a  purple-red  coloration.  In  the  presence  of  cane-sugar  and  sulphuric 
acid,  they  yield  a  purple  coloration  immediately.  This  reaction  is 
attributable  to  the  sphingosine  radical. 

Cerebrosides  are  absent  in  the  brains  of  fetal  animals,  but  with  the 
advance  of  medullation  they  appear  in  abundance.  It  is  therefore 
assumed  that  the  cerebrosides  are  constituents  originating  in  medullary 
sheaths  rather  than  in  the  axons  or  nerve-cells. 


THE   CARBOHYDRATE  ESTERS. 

Phosphoric  acid  esters  of  d-glucose  and  d-ribose  occur  among  the 
products  of  the  partial  hydrolysis  of  nucleic  acids.  They  will  fall  under 
more  extended  consideration  in  a  later  chapter. 

Sulphuric  acid  esters,  the  Glucothionic  Acids  have  been  found  by 
Levene  and  Mandel  in  a  variety  of  animal  tissues,  the  nature  of  the 
carbohydrate  radical  is  not  yet  established. 

A  sulphuric  acid  ester  of  an  amino-polysaccharide,  Chondroitin- 
Sulphuric  Acid,  CigH^NOisHSCX  occurs  in  important  amounts  in  bones 
and  other  sclerous  tissues  and  also  in  the  walls  of  the  great  arteries 
and  in  certain  pathological  tissues.  It  is  a  normal  constituent  of  urine 
in  very  small  amounts.  It  is  soluble  in  water,  yielding  levorotatory 


92     DISACCHARIDES,  POLYSACCHARIDES  AND  GLUCOSIDES 

solutions,  and  is  precipitable  from  aqueous  solutions  by  alcohol. 
Hydrolysis  by  dilute  hydrochloric  acid  yields  sulphuric  acid  and 
Chondroitin: 


+     H20  Ci8H27NOi4     +     H2SO4 

chondroitin  sulphuric  acid       +     water      =     ohondroitin         -f-     sulphuric  acid. 

Chondroitin  reduces  Fehling's  solution.  On  further  hydrolysis  it 
yields  d-galactose  and  d-glucuronic  acid;  it  appears  to  be  a  compound 
of  glucuronic  acid  and  an  amino-hexose,  Chondrosamin,  or  amino- 
galactose. 

REFERENCES. 
GENERAL: 

Armstrong:     Simple  Carbohydrates  and  the  Glucosides.     London.     2d  edition. 
SYMBIOSIS: 

Schottelius:     Arch.  f.  Hygiene,  1898,  34,  p.  210;  1902,  42,  p.  48;  1908,  67,  p.  177. 

Nuttal  and  TUerf  elder:     Zeit.  f.  physiol.  Chem.,  1895,  21,  p.  109;  1896,  22,  p.  62. 

Armsby:     U.  S.  Bureau  of  Animal  Industry,  Bull.  139,   1911. 

McCollum  and  Davis:     Jour.  Biol.  Chem.,  1915,  20,  p.  641. 

Kuriyama  and  Mendel:     Ibid.,  1917,  31,  p.  125. 
AMINOPOLYSACCHARIDES  AND  CARBOHYDRATE  ESTERS: 

Alsberg  and  Hedblom:     Jour.  Biol.  Chem.,  1909,  6,  p.  483. 

Morgulis:     Am.  Jour.  Physiol.,   1917,  43,  p.  328  (chitin). 

Mandel  and  Levene:     Zeit.  f.  physiol.   Chem.,    1905,  45,   p.   386;  Biochem.   Zeit., 
1907,  4,  p.  78. 

Levene  and  La  Forge:     Jour.  Biol.  Chem.,  1913,  15,  p.  155;  1914,  18,  pp.  123  and 
237;  1915,  20,  p.  433. 

Levene:     Ibid.,   1916,  26,  p.   143. 

Levene  and  Lopez-Sudrez:     Ibid.,  1916,  25,  p.  511;  1916,  26,  p.  373. 

Levene:     Ibid.,  1917,  31,  p.  609. 


CHAPTER  V. 

THE    HYDROAROMATIC    DERIVATIVES:    THE    CYCLOSES, 
CHOLESTEROL  AND  CHOLIC  ACID. 

GENERAL  CHARACTERISTICS. 

A  class  of  bodies  here  claims  our  consideration,  the  members  of 
which,  while  chemically  distinct,  are,  in  their  physical  behavior  and 
physiological  properties  intermediate  in  character  between  the  carbo- 
hydrates and  the  fats.  At  the  one  extremity  we  have  the  cy closes, 
which  although  polyatomic  alcohols,  nevertheless  resemble  sugars  in 
their  solubility  in  water,  their  percentage-composition  which  is  repre- 
sented by  the  formula  CeH^Oe,  and  their  decidedly  sweet  taste.  At  the 
other  we  have  cholesterol  and  the  cholesterol  esters  or  waxes  which 
resemble  the  fats  in  their  insolubility  in  water  and  solubility  in  organic 
solvents,  and  which  are  constantly  associated  with  fats  and  fatty 
substances  in  the  tissues  in  which  they  occur.  They  all  contain  a 
reduced  benzole-ring  and  are  thus  related  to  the  Terpenes;  they  are 
furthermore  hydroxy-derivatives  and  thus  yield  a  variety  of  color- 
reactions  which  depend  upon  the  presence  of  a  hydroxyl  radical  in  the 
benzole -ring. 

The  extreme  importance  of  these  substances  in  the  life  of  tissues  has 
only  very  recently  come  to  be  suspected,  but  the  variety  of  parts  they 
are  now  known  to  play  in  essential  activities  of  the  living  cell  is  so 
extensive  that  we  have  come  to  regard  them  as  constituting  a  very 
significant  factor  indeed  in  the  life-economy.  Thus  Inosite  in  combina- 
tion with  phosphoric  acid  is  an  important  constituent  of  seeds  and  the 
rapidly  growing  parts  of  plants,  while  in  animal  tissues  inosite  is  found 
in  a  variety  of  situations  and  forms  an  integral  part  of  the  molecule 
of  the  active  principle  of  the  anterior  lobe  of  the  Pituitary  Gland. 
Cholesterol  is  found  wherever  fats  occur  in  animal  tissues,  and  the 
remarkable  effects  which  it  exercises  upon  the  growth  of  epithelial 
tissues,1  show  that  it  plays  an  important  physiological  role.  Choles- 
terol esters  or  Waxes  occur  in  abundance  in  vegetable  tissues,  while  in 
mammals  they  occur  in  noteworthy  amounts  in  the  fatty  sheaths  of 
medullated  nerves,  and  in  the  cortex  of  the  Suprarenal  Gland.  Cholic 
Acid,  which  is  probably  a  derivative  of  cholesterol,  occurs  combined 
with  amino-acids  (amino-acetic  acid  or  ethyl  amino-sulphonic  acid) 
in  the  bile,  and  the  salts  which  these  acids  form  with  sodium,  play  an 
essential  part  in  accomplishing  the  digestion  and  assimilation  of  fats. 

1  Cf.  Chapter  xx. 


94  CYCLOSES,  CHOLESTEROL  AND  CHOLIC  ACID 

It  is  questionable  whether  animal  tissues  are  able  to  accomplish  the 
synthesis  of  any  of  these  substances;  in  fact  all  the  evidence  at  present 
available  contributes  to  show  that  they  cannot,  and  that  we  are 
absolutely  dependent  upon  vegetable  tissues  for  our  supplies  of  these 
very  essential  materials.  The  investigations  of  Gardner,  Denis 
Chalatov  and  Anistchakov  have  shown  that  addition  of  cholesterol  to 
the  dietary  in  abnormal  amounts,  increases  the  cholesterol-content  qf 
the  tissues,  while  a  diet  extremely  deficient  in  cholesterol-results  in  a 
like  deficiency  of  cholesterol  in  the  blood  and  tissues.  On  the  other 
hand,  in  vegetable  tissues  terpenes  and  terpene-derivatives  abound  so 
that  the  ultimate  source  of  cholesterol  in  the  diet  w^ould  appear  to 
reside  in  these  products  of  the  synthetic  activity  of  plants. 

The  power  of  the  animal  organism  to  destroy  cholesterol  is  very 
limited,  and  if  a  considerable  excess  be  administered  in  the  diet,  the 
unutilized  cholesterol  is  stored  away  in  various  tissues,  particularly 
in  the  liver,  spleen  and  suprarenal  bodies.  In  certain  animals,  for 
example  rabbits,  but  not  in  others,  the  excess  of  cholesterol  is  in  part 
deposited  in  the  interior  of  the  arterial  walls,  leading  to  the  formation 
of  lesions,  which  simulate  arteriosclerotic  lesions  of  the  arteries  in 
human  beings.  The  normal  channel  of  excretion  of  cholesterol  would 
appear  to  be  the  bile,  in  which  it  is  present  in  part  in  the  form  of  unal- 
tered cholesterol,  and  in  part  in  the  form  of  cholic  acid,  combined  with 
amino-acetic  acid  or  amino-ethyl-sulphonic  acid  to  form  the  "bile- 
acids."  Both  of  these  substances  are  in  part  reabsorbed  from  the 
intestine,  so  that  there  is  a  tendency  for  cholesterol  and  its  products 
to  circulate  in  the  body,  and  accumulate  in  the  tissues.  Of  course  this 
process  cannot  go  on  unchecked,  otherwise  the  accumulations  of 
cholesterol  in  the  tissues  would  soon  extinguish  their  functional 
activities.  It  appears  possible  from  the  abundance  of  cholesterol 
esters  in  the  suprarenal  cortex,  particularly  during  cholesterol  over- 
feeding, that  the  suprarenal  glands  may  play  a  part  in  assisting  to 
eliminate  or  destroy  cholesterol,  but  regarding  the  nature  of  the 
ultimate  products  which  may  be  formed  in  this  process  we  are  entirely 
in  the  dark.  Inosite,  on  the  other  hand,  which  contains  within  itself 
a  much  higher  proportion  of  oxygen  than  cholesterol,  is  partially 
oxidized  by  animal  tissues  and  the  products  of  its  oxidation  appear  to 
be  indistinguishable  from  those  of  carbohydrate-metabolism. 

Not  even  inosite,  however,  and  still  less  cholesterol  are  of  importance 
from  the  purely  nutritive  aspect,  i.  e.,  as  sources  of  energy.  The 
calorific  value  of  the  hydro-aromatic  fraction  of  the  diet  is  so  small  as 
to  be  negligible  in  comparison  with  the  total.  Their  significance  lies 
elsewhere,  and  if  we  revert  to  the  analogy  of  inanimate  machines  we 
must  class  them  with  the  lubricants  and  other  accessory  substances 
which  are  essential  to  the  smooth  running  of  the  machine,  rather  than 
with  the  fuel  which  supplies  the  energy  of  the  machine.  Indirectly, 
indeed,  they  must  contribute  to  the  available  energy-value  of  the  diet 
by  permitting  its  more  efficient  consumption,  just  as  the  judicious 


CYC  LOSES  95 

employment  of  lubricants  will  diminish  the  necessary  consumption  of 
gasoline  in  an  automobile-engine.  Their  influence  upon  the  nutrition 
of  animals  is  indirect,  however,  and  not  direct,  and  the  hydro-aromatic 
derivatives  must  for  this  reason  be  classified  as  Accessory  Foodstuffs 
or  foodstuffs  which  are  primarily  utilized  for  other  purposes  than  the 
production  of  work  and  heat,  or  the  building  up  of  the  structural 
elements  of  tissues. 

THE   CYCLOSES. 

The  hydro-aromatic  compounds  which  lie  nearest  to  the  carbo- 
hydrates in  their  physical  properties  and  physiological  behavior  are 
the  Cy closes,  or  hexa-hydroxy-benzoles,  which  are  represented  by  the 
formula : 

CHOH 


HOHC      CHOH 


HOHC      CHOH 

\/ 

CHOH 

A  number  of  isomeric  compounds  are  represented  by  this  formula, 
differing  from  one  another  in  the  arrangement  of  hydrogen  and  hydroxyl 
groups  about  the  carbons.  The  form  which  occurs  in  animal  tissues  is 
optically  inactive,  the  levo-  and  dextrorotatory  carbons  being  balanced 
and  equalized  within  the  molecule.  This  cyclose  is  designated  Inosite. 
In  vegetable  tissues  it  is  found  widely  distributed,  occasionally  in  the 
form  of  ester-like  compounds  (dambonite,  bornesite),  but  chiefly  in 
the  form  of  the  hexaphosphate,  the  calcium-magnesium  salt  of  which 
is  known  commercially  as  Phytin.  This  substance  occurs  particularly 
abundantly  in  seeds  and  grains,  the  husks  of  which  also  contain  a  fer- 
ment, Phytase,  which  is  capable  of  splitting  the  compound,  in  aqueous 
solution,  into  inosite  and  phosphoric  acid,  a  hydrolysis  which  other- 
wise can  only  be  accomplished  completely  by  exposing  the  substance 
in  acid  solution  to  a  temperature  very  considerably  above  that  of  boil- 
ing water.  Intermediate  steps  in  the  hydrolysis  of  inosite  hexaphos- 
phate by  phytase  are  the  tri-  and  mono-phosphates  which  do  not, 
however,  occur  preformed  in  the  tissues  of  grains. 

In  mammals  i-inosite  is  found  in  small  amounts  in  muscular  tissue, 
from  which  it  was  first  obtained  and  recognized  as  a  distinct  chemical 
entity.  It  is  also  found  in  combination  with  a  complex  fatty  substance, 
containing  phosphorus  and  nitrogen,  in  the  tissue  of  the  anterior  lobe 
of  the  pituitary  body.  This  compound,  to  which  the  name  Tethelin 
has  been  applied,  is  probably  the  physiologically  active  principle  of  the 
gland.  On  somewhat  prolonged  hydrolysis  by  alkalies  and  acids  the 
substance  breaks  up  and  yields  free  i-inosite. 

Inosite  is  readilv  soluble  in  water  and  alcohol  and  is  obtained  in  the 


96  CYCLOSES,  CHOLESTEROL  AND  CHOLIC  ACID 

form  of  fine  white  acicular  crystals  by  the  addition  of  ether  to  an 
alcoholic  solution.  It  has  a  sweet  taste,  but  being  neither  actually  nor 
potentially  an  aldehyde  or  ketone,  it  does  not  reduce  metallic  oxides 
in  alkaline  solution,  and  hence,  of  course,  does  not  reduce  Fehling's 
solution.  It  is  precipitated  from  aqueous  solutions  by  lead  acetate 
containing  an  excess  of  lead  oxide  ("basic  lead  acetate"). 

Inosite  may  be  recognized  by  the  above  peculiarities,  by  its  melting- 
point  (225°),  and  by  the  following  characteristic  reactions: 

Gallois'  Reaction. — A  drop  of  inosite  solution  is  mixed  with  a  drop  of 
mercuric  nitrate  solution  and  heat  gently  applied  until  the  water  has 
evaporated.  A  yellow  color  at  first  appears  which  changes  on  further 
heating  to  a  deep  red.  This  color  disappears  on  cooling,  and  reappears 
on  reheating. 

Scherer's  Reaction. — A  few  crystals  of  inosite  are  dissolved  in  a  drop 
or  two  of  nitric  acid  of  specific  gravity  1.2,  and  an  equal  volume  of  ten 
per  cent,  calcium  chloride  solution  is  added  and  the  same  volume  of  a 
one  per  cent,  solution  of  platinic  chloride.  This  mixture  is  evaporated 
to  dryness  and  the  residue  heated,  when  a  rose-red  color  appears,  which 
disappears  on  cooling,  and  reappears  with  a  bluish  nuance  on  reheating. 

Inosite  is  found  in  small  amounts  in  normal  urine,  and  the  amount 
increases  in  certain  pathological  conditions,  particularly  in  diabetes 
insipidus  and  in  Bright's  disease.  The  administration  of  inosite  in 
unusual  amounts  by  mouth  gives  rise  to  transient  diarrhoea  and  to  an 
increase  in  the  Creatinine  output  in  the  urine,  a  fact  which,  in  the  light 
of  considerations  which  will  be  detailed  in  subsequent  chapters,  may 
possibly  indicate  increased  destruction  of  tissue-substances.  Only  a 
very  small  proportion  of  the  inosite  administered  by  mouth  is  excreted 
in  the  urine,  the  remainder  being  oxidized  and  eliminated  in  the  form 
of  products  which  are  apparently  indistinguishable  from  those  of 
ordinary  carbohydrate-combustion.  In  phloridzinized  dogs  the  excre- 
tion of  d-glucose  in  the  urine,  already  a  maximum,  is  increased  by 
administration  of  inosite,  and  if  the  ^additional  output  of  glucose  be 
added  to  the  inosite  which  is  excreted  unchanged  in  the  urine,  the  sum 
is  approximately  equal  to  the  inosite  administered.  Under  these 
circumstances,  therefore,  the  ring-formation  appears  to  undergo  a 
simple  splitting  with  the  partial  transformation  of  inosite,  molecule 
for  molecule,  into  glucose. 

Cycloses  other  than  i-inosite  occur  in  vegetable  tissues  but  with  one 
exception  have  not  as  yet  been  identified  among  the  constituents  of 
animal  tissues.  The  exception  is  Scyllite  which  is  found  in  the  tissues 
of  the  bony  (Teleost)  fishes.  It  gives  Scherer's  reaction  and  is  optically 
inactive,  but  it  may  be  distinguished  from  i-inosite  by  its  very  high 
melting-point;  380°  as  contrasted  with  225°. 

In  vegetable  tissues  occur  1-inosite  in  the  form  of  the  methyl  ester 
in  quebracho  bark,  d-1-inosite  or  racemic  inosite  (a  mixture  of  the  d-  and 
1-varieties)  in  the  leaves  of  mistletoe,  and  d-inosite  in  the  rosin  and 
needles  of  conifers,  in  senna  leaves  and  in  India-rubber. 


CHOLESTEROL  AND  THE  PHYTOSTEROLS  97 

CHOLESTEROL    AND    THE   PHYTOSTEROLS. 

Cholesterol,  C27H45OH,  may  be  represented  so  far  as  our  knowledge 
at  present  extends,  by  the  formula  of  von  Fiirth: 


CH3 


CH3 


HOHC 


it  is  found  in  all  animal  fats  or  oils,  in  small  quantities,  in  bile,  blood, 
milk,  yolk  of  egg,  the  medullated  sheaths  of  nerve-fibers,  the  liver, 
kidneys  and  suprarenal  bodies.  It  is  contained  in  considerable  amount 
in  cod-liver  oil.  Under  pathological  conditions  it  is  found  to  constitute 
a  very  large  proportion  of  the  most  frequently  occurring  type  of  gall- 
stones, the  conditions  which  ordinarily  hold  cholesterol  in  solution  in 
bile,  being  in  these  cases,  it  appears,  deficient.  It  occurs  also  in 
atheromata  of  the  arteries,  in  tubercular  cysts  and  in  carcinomatous 
tissue. 

When  precipitated  from  alcoholic  solution  by  the  addition  of  water, 
or  when  deposited  in  the  body,  as  in  gall-stones,  cholesterol  forms 
characteristic  crystals  with  one  re-entrant  angle,  resembling  flat 
rectangular  plates  with  one  corner  knocked  out  (Fig.  2).  These 
crystals  contain  one  molecule  of  water  and  are  white,  of  a  waxy  con- 
sistency, insoluble  in  water,  soluble  in  alcohol,  ether,  benzol,  etc.,  and 
in  fatty  oils.  When  crystallized  from  anhydrous  alcohol-ether  mixtures 
cholesterol  forms  acicular  crystals  without  any  water  of  crystallization. 
Cholesterol  may  be  held  in  solution  or  suspended  in  emulsified  form  in 
water  by  the  addition  of  soaps,  saponins,  bile-salts,  or  lecithin,  and  it  is 
by  this  means  that  it  is  held  suspended  in  the  bile  and  other  tissue- 
fluids. 

As  has  been  stated  above,  there  is  reason  to  suppose  that  cholesterol 
may  possibly  be  decomposed  in  the  suprarenal  glands,  and  a  portion  is 
possibly  converted  into  cholic  acid  in  the  liver,  but  for  the  rest,  so  far 
as  we  know  at  present,  the  main  channel  of  excretion  for  cholesterol 
is  the  bile.  The  cholesterol  which  thus  finds  its  way  into  the  upper 
part  of  the  small  intestine,  along  with  the  cholesterol  of  the  food,  is  in 
part  reabsorbed  and  in  part  retained  in  the  intestine  until  it  is  voided 
7 


98 


CYCLOSES,  CHOLESTEROL  AND  CHOLIC  ACID 


with  the  feces.  This  latter  portion  of  the  cholesterol  becomes  subject 
in  the  lower  intestine  to  the  putrefactive  action  of  bacteria,  which 
results  in  its  reduction  to  a  derivative  of  cholesterol  designated 
Coprosterol,  containing  two  additional  hydrogen  atoms,  and  represented 
by  the  formula  C27H47OH. 

This  inefficient  method  of  excretion  would  lead  undoubtedly  to  a 
continual  accumulation  of  cholesterol  within  the  tissues,  if  it  were  not 
assisted  by  some  means  of  destruction  of  the  accumulated  excess. 
The  power  of  the  body  to  destroy  cholesterol  is,  however,  very  limited, 
and  if  cholesterol  be  administered  in  the  dietary  in  unusual  quantities, 
it  forms  deposits  in  various  organs,  notably  the  liver  and  suprarenal 
glands,  and  may  ultimately  lead  to  the  formation  of  serious  lesions. 
There  is  therefore,  under  ordinary  circumstances,  rather  a  delicate 
balance  between  the  intake  of  cholesterol  in  the  food  on  the  one  hand, 


FIG.  2. — Cholesterol  crystals.     (After  Hawk.) 

and  its  output  in  the  feces,  and  destruction  in  the  tissues  on  the  other. 
If  the  power  of  the  tissues  to  destroy  or  alter  cholesterol  is  diminished 
for  any  reason  we  may  anticipate  that  the  excretory  apparatus  will 
be  found  inadequate,  and  that  cholesterol  will  accumulate  in  the  body. 
It  is  to  this  that  we  must  probably  attribute  the  accumulation  of 
cholesterol  which  has  been  observed  by  Wacker  in  the  subcutaneous 
fatty  tissues  of  aged  people,  the  decline  in  the  activity  of  the  tissues 
which  accompanies  age  probably  resulting  in  a  deficient  power  of 
destroying  cholesterol.  It  has  been  observed  by  Luden  that  the 
cholesterol-content  of  the  blood  in  carcinomatous  patients  is  usually 
high  and  that  oxidation-products  of  cholesterol  which  are  present  in 
normal  blood  are  frequently  absent  in  these  cases. 

The  administration  of  unusual  amounts  of  cholesterol  to  young 
animals  results  in  marked  effects  upon  their  Growth,  which  will  be  fully 
discussed  in  a  later  chapter.  If  cholesterol  be  administered  to  animals 


CHOLESTEROL  AND  THE  PHYTOSTEROLS        99 

(rats)  inoculated  with  carcinomatous  tissue,  the  cancer  grows  much 
more  rapidly  than  in  normal  animals  and  "metastases"  or  fresh  growths 
in  localities  distant  from  the  site  of  the  primary  growth,  are  formed 
much  more  numerously  and  in  a  much  higher  proportion  of  animals. 
In  this  connection  it  is  significant  to  observe  that  Carcinoma  is  primarily 
a  disease  of  old  age  so  far  as  manifest  growth  or  accretion  of  the  parasitic 
tissue  is  concerned.  It  very  rarely  manifests  itself  in  man  before  thirty 
and  increases  in  frequency  very  decidedly  with  advancing  age,  the 
incidence  between  the  ages  of  sixty-five  and  seventy-five  being  no  less 
than  ten  times  as  great  as  between  thirty-five  and  forty-five.  It 
is,  however,  impossible  to  initiate  carcinomatous  growths  in  animals 
by  administration  of  cholesterol,  unless  carcinoma-tissue  is  already 
present  as  a  result  of  inoculation  or  spontaneous  development,  so  that 
cholesterol  cannot  be  looked  upon  as  a  cause,  but  rather  as  a  favoring 
condition  of  cancer-growth.  It  must  be  remembered  that  our  estimate  of 
the  age  of  incidence  of  carcinoma  is  founded  upon  the  date  at  which  the 
growth  obtrudes  itself  upon  the  attention  of  the  patient  or  physician. 
For  how  long  prior  to  this  its  beginnings  have  been  actually  resident  in 
the  body,  we  have  no  means  of  estimating,  but  judging  by  the  analogy 
afforded  by  other  growth-phenomena  (cf .  Chapter  XX)  we  may  infer 
that  the  date  of  origin  of  the  growth  probably  precedes  by  a  consider- 
able interval  the  date  of  its  obvious  manifestation,  so  that  despite  the 
fact  that  cholesterol  cannot  initiate  cancer,  the  date  of  its  diagnosis, 
and  therefore  its  "apparent"  or  "statistical"  date  of  incidence  may  very 
possibly  be  determined  by  the  acceleration  of  its  growth  due  to  an 
accumulation  of  cholesterol  in  the  tissues. 

Cholesterol  yields  the  following  series  of  color  reactions  together 
with  others,  for  description  of  which  the  student  is  referred  to  special 
monographs : 

Salkowski's  Eeaction. — Cholesterol  is  dissolved  in  chloroform  and  an 
equal  volume  of  concentrated  sulphuric  acid  is  added.  The  solution 
is  colored  blood-red  which  changes  gradually  to  purple.  If  the  mixture 
is  poured  out  in  a  shallow  layer  and  exposed  to  the  air,  the  purple 
changes  to  blue,  then  green  and  ultimately  yellow. 

Liebermann-Burchard  Reaction. — Cholesterol  is  dissolved  in  a  small 
amount  of  chloroform  in  a  dry  test-tube,  a  few  drops  of  acetic  anhydride 
are  added  and  then  concentrated  sulphuric  acid  is  added  drop  by  drop. 
The  mixture  becomes  red,  then  blue  and  finally,  if  not  too  much 
cholesterol  and  sulphuric  acid  have  been  added,  a  permanent  green. 

Obermuller's  Reaction. — Dry  cholesterol  is  heated  in  a  glass  tube  with 
two  or  three  drops  of  propionic  anhydride  until  it  melts.  On  cooling 
the  mass  turns  first  violet,  then  blue,  green,  orange,  and  finally  red. 

Schiff' s  Reaction. — To  dry  cholesterol  in  an  evaporating  dish  add  a 
trace  of  ferric  chloride,  strong  hydrochloric  acid  and  chloroform,  and 
evaporate  the  mixture  nearly  to  dryness,  when  the  edge  of  the  residue 
begins  to  turn  violet.  Then  add  more  chloroform,  evaporate  to  dry- 
ness  and  heat.  The  whole  mass  turns  violet  first  with  a  reddish  and 
later  with  a  bluish  nuance,  and  finally  a  dirty  green. 


100  CYCLOSES,  CHOLESTEROL  AND  CHOLIC  ACID 

Neuberg-Rauchwerger's  Reaction. — This  reaction  is  of  exceptional 
interest  because  it  is  also  given  by  the  bile-acids  and  certain  other 
derivatives  of  the  terpenes.  A  common  origin  of  the  bile-acids  (cholic 
acid)  and  cholesterol  is  thus  indicated.  With  rhamnose  or  better  still, 
with  d-methyl-furfurol  and  concentrated  sulphuric  acid,  an  alcoholic 
solution  of  cholesterol  gives  a  pink  ring,  or  after  mixing  the  two  liquids 
and  cooling,  a  pink  solution. 

Lifschutz's  Reaction. — Dissolve  a  few  milligrammes  of  cholesterol 
in  two  c.c.  of  glacial  acetic  acid,  add  a  few  drops  of  benzoyl  superoxide, 
and  boil.  On  adding  four  drops  of  concentrated  sulphuric  acid  to  the 
solution  a  green  coloration  is  obtained,  which  rapidly  changes  to  violet, 
then  to  blue.  Oxidation-products  of  cholesterol  yield  this  reaction 
without  preliminary  treatment  with  benzoyl  superoxide,  and  in  this 
way  oxidation-products  of  cholesterol  have  been  detected  in  the  blood 
and  tissues,  and  especially  in  cholesterol-concretions  (gall-stones)  in  the 
gall-bladder. 

In  plant-tissues  there  are  found  a  variety  of  substances,  the  Phyto- 
sterols,  which  are  more  or  less  closely  allied  to  cholesterol.  The  best, 
known  of  these  is  Sitosterol,  an  isomer  of  cholesterol,  which  occurs  in 
wheat,  rye,  linseed-oil  and  the  calabar  bean.  It  differs  from  choles- 
terol in  crystalline  form,  melting-point  (137°  contrasted  with  148.5°  for 
cholesterol)  and  optical  rotatory  power.  Its  solubilities  in  various 
organic  solvents,  and  the  color  reactions  which  it  yields  are  similar 
to  those  of  cholesterol.  It  is  absorbed  together  with  cholesterol  from 
the  intestine. 

In  fungi  a  series  of  phytosterols  are  found  which  contain  a  smaller 
proportion  of  hydrogen  than  cholesterol,  and  furthermore,  differ  from 
cholesterol  in  not  yielding  Salkowski's  reaction. 


BILE-CONCRETIONS;   AMBERGRIS. 

The  concretions  which  occasionally  form  in  the  gall-bladder  are  of 
three  types,  formed  respectively  of  Calcium  Carbonate,  Bile-pigments 
and  Cholesterol,  Each  of  these  types  of  gall-stones  is  usually  con- 
taminated with  a  larger  or  smaller  proportion  of  the  constituents  of  the 
other  types.  The  cholesterol-stones  have  a  waxy  glistening  and  cry- 
stalline fracture,  and  are  frequently  deposited  in  concentric  layers. 
They  are  often  facetted  by  the  pressure  of  adjacent  stones,  while  their 
color  is  sometimes  white,  but  more  frequently  tinged  with  bile-pigments. 

The  cholesterol-stones  are  the  type  which  most  frequently  occur  in 
man.  The  conditions  leading  to  their  formation  are  unknown  but  it  is 
parhaps  a  significant  fact,  in  view  of  the  accumulation  of  cholesterol  in 
the  tissues  with  advancing  age,  that  the  incidence  of  cholelithiasis 
increases  progressively  with  the  advance  of  years,  over  75  per  cent,  of 
cases  occurring  in  persons  over  forty  years  of  age.  It  is  furthermore 
stated  that  cholelithiasis  is  more  frequent  in  carcinomatous  than  in 
non-carcinomatous  subjects. 


CHOLESTEROL  ESTERS       : 


On  the  other  hand  the  deposition  of  cholesterol  may  frequently 
originate,  not  so  much  in  the  abundance  of  this  substance  in  the  bile, 
as  in  its  diminished  solubility  therein.  An  increase  in  the  albumin- 
content  of  bile,  as  in  inflammatory  conditions,  or  by  the  addition  of 
egg-albumin  to  bile  in  vitro  may  lead  to  the  deposition  of  cholesterol 
and  it  is  stated  that  certain  bacteria,  particularly  the  typhoid  bacillus, 
diminish  the  solubility  of  cholesterol  in  bile  which  they  inhabit. 

The  proportion  of  cholesterol  in  cholesterol-stones  varies  from 
sixty-four  to  ninety-eight  per  cent.  In  addition  there  occur  derivatives 
of  cholesterol  which  yield  Lifschiitz's  reaction  without  preliminary 
oxidation,  and  are  probably,  therefore,  derivatives  originating  from 
cholesterol  by  oxidation.  Similar  substances  are  found  in  the  blood 
of  normal  persons,  but  are  deficient  in  or  absent  from  the  blood  of 
persons  afflicted  with  carcinoma  (Luden). 

The  biliary  concretions  of  the  sperm  whale  (Physeter  macrocephalw) 
are  occasionally  found  floating  upon  the  sea,  or  cast  up  upon  the  shores 
of  oceans  inhabited  by  these  mammals.  They  are  found  in  dull  gray  or 
black  masses,  having  a  peculiar  sweet  earthy  odor,  and  form  the 
Ambergris  of  commerce.  When  taken  directly  from  the  intestinal 
canal  of  whales  it  is  of  a  deep  gray  color,  soft  consistence  and  disagree- 
able odor,  but  on  exposure  to  air,  it  hardens  and  acquires  the  charac- 
teristic odor  just  described.  Ambergris  formerly  enjoyed  a  high 
reputation  as  a  therapeutic  agent  but  its  therapeutic  virtues  probably 
resided  in  its  scarcity  and  expensiveness.  At  the  present  time 
ambergris  is  of  importance  solely  in  the  manufacture  of  perfume  in 
which  its  utility  depends  upon  the  rather  extraordinary  property  it 
possesses,  when  added  to  perfumes  in  minute  amounts,  of  very  markedly 
enhancing  their  "floral"  fragrance. 

Ambergris  consists  in  the  main,  frequently  to  the  extent  of  eighty- 
five  per  cent.,  of  a  substance,  Ambrine,  which  very  closely  resembles 
cholesterol  in  its  solubilities,  general  appearance  and  composition.  It 
is  insoluble  hi  water,  highly  soluble  in  alcohol,  ether  and  oils,  and 
crystallizes  in  white  shining  plates. 

CHOLESTEROL  ESTERS. 

Cholesterol  esters  of  the  fatty  acids  are  very  widely  distributed  in  the 
vegetable  kingdom.  In  the  animal  kingdom  they  are  found  in  the 
blood  and  lymph,  in  the  medullated  sheaths  of  nerves,  in  the  cortical 
tissues  of  the  suprarenal-  gland  and  in  the  secretions  of  the  sebaceous 
glands.  The  so-called  fat  or  grease  of  sheeps'  wool,  which,  when  refined 
is  commercially  known  as  "Lanoline,"  consists  almost  entirely  of  a 
mixture  of  the  palmitate,  oleate  and  stearate  of  cholesterol  together 
with  a  variable  proportion  of  water. 

The  fatty  acid  esters  of  cholesterol  resemble  the  true  fats,  or  fatty 
acid  esters  of  glycerol,  in  their  solubility  in  organic  solvents,  and 
insolubility  in  water.  They  differ,  however,  from  the  fats  in  the 


,162  -:  :*:  :cy$LO&E&  CHOLESTEROL  AND  CHOLIC  ACID 

comparative  difficulty  with  which  they  are  hydrolyzed  or  "saponified" 
by  alkalies,  in  their  resistance  to  the  action  of  bacteria,  so  that  they  do 
not  become  "rancid,"  and  in  the  property  they  possess  of  absorbing 
or  mechanically  imbibing  a  large  proportion  of  water  to  form  a  mass 
which  still  retains  a  fatty  consistency.  For  this  reason  lanoline  has 
of  late  come  to  be  employed  very  widely  in  therapeutics  as  a  vehicle 
for  aqueous  solutions  of  drugs  which,  through  this  agency,  may  be 
applied  as  salves. 

The  cholesterol  esters  differ  from  cholesterol  is  not  being  emulsi- 
fiable  in  water  containing  soaps.  Acetyl  cholesterol  C^yELs.OOC.CHs 
is  also  devoid  of  the  characteristic  action  of  cholesterol  upon  the 
growth  of  carcinomata.  It  would  seem  unlikely  that  this  is  due  merely 
to  the  replacement  of  a  hydroxyl-group  by  an  acetyl-group,  more 
especially  since  a  variety  of  soluble  and  insoluble  hydroxyl-derivatives 
of  hydroaromatic  substances  have  been  found  to  be  devoid  of  action 
upon  the  growth  of  carcinoma.  It  appears  more  likely  that  the  loss 
of  emulsifiability  consequent  upon  the  replacement  of  the  hydroxyl- 
group  prevents  the  distribution  of  acetyl  cholesterol  by  the  blood  and 
tissue-fluids  to  the  cells  of  the  carcinomatous  tissue. 

The  cholesterol  esters  are  of  exceptional  interest  to  the  physical 
chemist  because  they  are  the  substances  which  were  first  observed  by 
Lehmann  to  display  the  remarkable  phenomenon  of  "fluid  crystals" 
or  drops  which,  while  spherical  and  retaining  the  characteristics  of 
fluids,  nevertheless  display  evidence,  afforded  by  the  unequal  refrac- 
tion of  light  in  different  axes,  of  the  possession  of  an  internal  crystalline 
structure.  While  other  bodies  are  now  known  which  display  this 
peculiarity,  the  cholesterol  esters  still  constitute  a  group  which  is  pre- 
eminently suitable  for  the  investigation  of  fluid  crystals.  They  may 
be  very  readily  obtained  by  gently  heating  the  esters  on  a  microscope 
slide  until  somewhat  above  the  melting-point,  and  allowing  to  cool  to  a 
little  above  the  melting-point. 

According  to  Adami  fluid  crystals,  presumably  containing  choles- 
terol esters,  may  be  observed  in  the  myelin  droplets  which  form  during 
the  degeneration  of  the  fatty  sheaths  of  medullated  nerves. 

THE  BILE-SALTS   AND  CHOLIC  ACID. 

The  mixed  bile-salts,  sodium  glycocholate  and  sodium  taurocholate 
may  readily  be  obtained  from  ox-bile  by  mixing  the  bile  with  animal 
charcoal,  evaporating  to  dryness,  extracting  with  hot  alcohol  and  add- 
ing ether  to  the  cooled  extract.  If  the  process  has  been  properly  per- 
formed, a  snow-white  precipitate  of  fine  acicular  crystals  ("Plattner's 
crystallized  bile")  is  obtained  which,  in  one  or  two  crystallizations, 
may  be  almost  freed  from  contamination.  The  two  salts  may  be 
separated  by  adding  lead  acetate  to  their  aqueous  solution,  by  which 
means  the  glycocholate  is  precipitated,  while  the  ta  irocholate 
remains  in  solution.  Sodium  glycocholate  is  the  most  abundant 


BILE-SALTS  AND  CHOLIC  ACID  103 

constituent  of  the  bile-salts  in  herbivorous  animals  and  in  man,  but  is 
absent  from  the  bile  of  carnivorous  animals.  Sodium  taurocholate, 
on  the  contrary,  only  occurs  in  small  amounts  in  the  bile  of  herbivora 
and  man,  while  it  is  abundant  in  the  bile  of  carnivora. 

The  bile-salts  are  readily  soluble  in  water,  yielding  solutions  which, 
like  solutions  of  the  saponins,  have  a  very  low  surface-tension,  foam 
readily,  and  hold  otherwise  insoluble  substances  in  solution  or  suspen- 
sion. This  is  especially  true  of  the  lecithins,  which  are  very  readily 
emulsified  by  bile-salts.  The  low  surface-tension  of  these  solutions 
is  utilized  very  frequently  in  manometers  for  measuring  very  minute 
changes  of  gas-pressure.  The  solution  of  bile-salts  does  not  "stick" 
or  form  drops  on  the  sides  of  the  containing  tube  as  water  frequently 
does,  and  the  meniscus  or  surface  of  the  fluid  is  flatter  than  that  of 
water,  enabling  a  reading  of  the  height  of  a  column  to  be  made  with 
greater  ease  and  accuracy. 

The  bile-salts  and  the  free  acids  are  further  characterized  by  the 
peculiar  taste  of  their  solutions,  at  once  bitter  and  sweet.  The  dry 
salts  form  a  very  fine  light  powder  which  is  irritating  when  it  comes 
into  contact  with  the  nasal  mucous  membranes. 

Hydrolysis  of  glycocholic  acid  by  boiling  with  barium  hydroxide 
yields  Glycocoll  and  Cholic  Acid: 

C23H39O3.CO.HN.CH2.COOH  +  H2O   =  C^H^Os.COOH  +  CH2NH2COOH 
glycocholic  acid  +     water      =     cholic  acid       +      ammo-acetic  acid. 

while  hydrolysis  of  taurocholic  acid  yields  Cholic  Acid  and  amino-ethyl 
sulphonic  acid  (Taurin)  : 


C23H39O3.CO.HN.CH2.CH2SO2.OH  +  H2O  =  C23H39O3COOH    +  H2N.CH2.CH2.SO2OH 
taurocholic  acid       +         water      =       cholic  acid     +     amino-ethyl-sulphonic  acid. 

The  characteristic  peculiarities  of  the  bile-acids  are  determined 
by  their  common  radical,  the  cholic-acid  fraction.  Free  cholic  acid,  is 
almost  insoluble  in  water,  but  its  salts  readily  dissolve,  forming  bitter- 
sweet solutions  which  are  dextrorotatory.  The  alkali  salts  of  cholic 
acid,  on  the  other  hand,  are  only  sparingly  soluble  in  alcohol,  while  the 
free  acid  dissolves  readily  in  this  solvent.  Cholic  acid  may  also  be 
recognized  by  the  following  characteristic  reactions: 

Hammarsten's  Reaction.  —  If  finely  powdered  cholic  acid  be  added  to  a 
twenty-five  per  cent,  solution  of  hydrochloric  acid,  a  violet-blue  colora- 
tion slowly  appears  which  gradually  changes  through  green  to  yellow. 

Mylius'  Reaction.  —  If  an  alcoholic  (about  five  per  cent.)  solution  of 
cholic  acid  in  alcohol  be  mixed  with  two  volumes  of  y^  iodine  solution 
in  alcohol,  and  the  mixture  slowly  diluted  with  water,  microscopic 
needles  of  an  iodine  addition-product  are  formed  which  are  blue  by- 
transmitted  light.  This  reaction  is  characteristic  for  cholic  acid  and  is 
not  given  by  the  conjugated  bile-acids  (glycocholic  or  taurocholic  acids). 

Pettenkofer's  Reaction.  —  With  a  little  cane-sugar,  on  careful  addition 
of  strong  sulphuric  acid,  it  yields  a  red  coloration.  This  reaction, 
which  is  probably  due  to  furfurol  formed  from  cane-sugar  by  the  action 


104 


CYCLOSES,  CHOLESTEROL  AND  CHOLIC  ACID 


of  the  sulphuric  acid,  is  not  absolutely  to  be  relied  upon,  since  similar 
reactions  (differing  from  one  another,  however,  in  the  absorption- 
spectra  of  the  fluids  produced)  are  yielded  by  a  variety  of  substances, 
for  example  proteins,  oleic  acid,  phospholipins,  amyl  alcohol  and 
morphine. 

Neuberg-Rauchwerger's  Reaction. — Also  given  by  cholesterol,  which 
see. 

The  structure  of  cholic  acid  has  not  yet  been  fully  elucidated,  but  it 
appears  to  be  definitely  established  that  it  is  a  derivative  of  the  hydro- 
aromatic  series.  The  decomposition-products  resulting  from  the 
variety  of  procedures  contain  fractions  which  are  closely  related  to 
products  which  are  similarly  obtained  from  other  hydro-aromatic 
derivatives,  such  as  cholesterol,  turpentine  and  camphor,  and  the 
identification  by  Panzer  of  hydroxy-hexahydro-phthalic  acid: 


OH 


H2C 


H2C 


\/ 
c 

/\ 


COOH 
/ 


\ 
CH2 


CHS 
/ 


CH 


COOH 


among  the  oxidation-products  of  cholic  acid  leaves  very  little  room  for 
doubt  that  hydro-aromatic  nuclei  exist  preformed  in  the  undecomposed 
molecule.  Apart  from  these  inferences,  however,  it  is  known  that 
cholic  acid  contains  one  carboxyl-group  and  two  primary  and  one 
secondary  alcohol-groups  united  to  a  hydrocarbon-complex  which 
contains  cyclic  linkages.  The  formula  may  therefore  be  written: 


C2oH 


fCHOH 
CH2OH 
31     CH2OH 
I  COOH 


A  related  acid  which  is  found  in  small  amounts  in  ox-bile  and  also  in 
gall-stones  is  Choleic  Acid,  which  differs  from  cholic  acid  in  its  per- 
centage composition  ^JLoCX)  and  in  its  relative  insolubility  in 
alcohol.  It  yields  a  blue  compound  with  iodine  and  also  gives  Ham- 
marsten's  reaction  with  hydrochloric  acid.  Desoxycholic  Acid,  how- 
ever, which  also  occurs  in  bile  and  in  gall-stones  yields  neither  of  these 
reactions  although  it  is  isomeric  with  choleic  acid. 


BILE-SALTS  AND  CHOLIC  ACID  105 

In  the  bile  of  animals  other  than  man  or  the  ox  are  found  a  variety 
of  acids,  which  have  as  yet  been  very  imperfectly  studied  but  which 
differ  in  composition  and  physical  characteristics  from  one  another 
and  from  cholic  acid.  A  common  origin  of  these  substances  is  probably 
to  be  sought  in  hydro-aromatic  radicals  contained  in  the  diet  and  derived 
ultimately  in  all  probability  from  vegetable  tissues. 

The  bile-salts  are,  in  part  at  least,  reabsorbed  from  the  intestine,  and 
bile-salts  administered  by  mouth  cause  a  remarkable  increase  in  the 
secretion  of  bile,  in  fact,  with  the  possible  exception  of  salicylic  acid, 
the  bile -salts  appear  to  be  the  only  true  Cholagogues  or  stimulants  of 
the  secretion  of  bile.1  When  they  are  injected  into  the  blood  or  forced 
into  the  blood  owing  to  an  obstruction  of  the  bile-ducts,  leading  to 
icterus  or  "jaundice,"  they  have  a  markedly  depressant  action  upon 
the  heart-muscle,  slowing  the  beat  very  decidedly,  and  in  large  amounts 
they  dissolve  the  red  blood-corpuscles  just  as  the  saponins  do.  Under 
these  circumstances  bile -salts  are  probably  excreted  in  part  in  the  urine, 
but  no  reliable  method  of  confirming  their  presence  in  the  urine  has 
yet  been  devised.  For  clinical  purposes  however,  this  is  not  an  incon- 
venience since  the  presence  of  bile  in  the  circulating  blood  is  always 
evidenced  by  the  appearance  of  Bile-pigments  in  the  urine  which  are 
readily  detected  in  a  variety  of  ways. 


REFERENCES. 

INOSITE: 

Starkenstein-'     Zeit  exp.  Path.  u.  Therp.,  1908-9,  5,  p.  378. 

Rose:     Biochem.  Bull.,  1912-13,  2,  p.  21. 

Anderson:     Jour.  Biol.  Chem.,  1912,  11,  p.  471;  1912,  12,  pp.  97  and  447;  1912- 

1913,  13,  p.  311;  1914,  17,  pp.  141,  151,  165,  171;  1914,  18,  pp.  425  and  441; 
1915,  20,  pp.  463,  475,  483,  493;  1916,  25,  p.  391. 

Anderson  and  Bosworth:     Ibid.,  1916,  25,  p.  399. 
CHOLESTEROL: 

von  Filrth:     Biochem.  Zeitschr.,   1915,  49,  p.  416. 
Bang:     Chemie  und  Biochemie  der  Lipoide.     Wiesbaden,  1911. 
Glikin:     Chemie  der  Fette,  Lipoide  und  Wachsarten.     Berlin,  1913. 
Lifschutz:     Zeit.  f.  physiol.  Chem.,  1909,  58,  p.  175;  1909,  63,  p.  222.     Biochem. 
Zeit.,  1913,  52,  p.  206.    Zeit.  f.  physiol.  Chem.,  1914,  91,  p.  309;  1914,  92,  p.  383; 

1914,  93,  p.  209. 

Doree  and  Gardner:    Proc.  Roy  Soc.  BM  1908,  80,  pp.  212  and  227;  1909,  81,  p.  109. 
Ellis  and  Gardner:     Ibid.,  1909,  81,  pp.  129  and  505;  1912,  84,  p.  461;  1912,  85,  p. 

385;  1913,  86,  p.  13. 

Fraser  and  Gardner:     Ibid.,  1909,  81,  p.  230;  1910,  82,  p.  559. 
Doree:     Biochem.  Jour.,   1909,  4,  p.   72. 
Gardner  and  Lander:    .Biochem.  Jour.,  1913,  7,  p.  576.     Proc.  Roy.  Soc.  B.,  1914, 

87,  p.  229. 
Chalatow:     Virchows  Arch.  Path.  u.  Anat.,  1912,  207,  p.  452.      Beitr.  Path.  Anat. 

u.  Allg.  Path.,  1914,  57,  p.  85. 
Anitschkow:     Beitr.  Path.  Anat.  u.  Allg.  Path.,  1913,  56,  p.  379;  1914,  57,  p.  201. 

Deutsch.  med.  Wchnschr.,  1913,  39,  p.  741. 

Weltmann  and  Biach:     Zeit.  exp.  Path.  u.  Therap.,  1913,  14,  p.  367. 
Bailey:     Proc.  Soc.  Exp.  Biol.  and  Med.,  1914,  12,  p.  68;  1915,  13,  p.  60. 

1  In  the  opinion  of  some  investigators,  however,  the  increase  in  the  secretion  of  bile 
which  results  from  the  administration  of  bile-salts  is  no  greater  than  that  which  would 
be  equivalent  to  the  amount  of  bile-salts  administered. 


106  CYCLOSES,  CHOLESTEROL  AND  CHOLIC  ACID 

RELATIONSHIP  OF  CHOLESTEROL  TO  CARCINOMA: 

Robertson  and  Burnett:     Jour.  Exp.  Med.,  1913,  17,  p.  344.     Proc.  Soc.  Exp.  Biol. 

and  Med.,  1913,  10,  pp.  140  and  143;  1913,  11,  p.  42;  1914,  12,  p.  33.     Jour,  of 

Cancer  Research,  1918,  3,  p.  75. 

Robertson  and  Ray:     Jour.  Biol.  Chem.,  1919,  37,  p.  443. 
Wacker:     Zeit.  f.  physiol.  Chem.,  1912,  80,  p.  383. 
Luden:     Jour.  Lab.  and  Clin.  Med.,  1916,  1,  p.  662;  1917,  3,  pp.  93  and  141;  1918, 

4,  p.  1.     Jour.  Biol.  Chem.,  1916,  27,  p.  273;  1917,  29,  p.  463. 
Sweet,  Corson,  White  and  Saxon:     Jour.  Biol.  Chem.,  1915,  21,  p.  309. 


CHAPTER  VI. 
THE  FATS. 

THE  TRUE  FATS. 

The  true  fats  are  compounds,  or  Esters  of  the  fatty  acids  with  the 
triatornic  alcohol  glycerol.1  Thus  tripalmitin  is  formed  by  the  union 
of  three  molecules  of  palmitic  acid  with  one  molecule  of  glycerol  and 
the  elimination  of  a  corresponding  number  of  molecules  of  water. 

CH2OH  HOOC.Ci5H31  CH2OOC.C15H3i 

CHOH          +         HOOC.C15H3i         =         CH.OOC.CuHn         +         3H2O 

CH2OH  HOOC.C15H3i  CH2OOC.Ci5H3i 

By  the  action  of  alkalies  this  process  is  reversed,  and  the  fatty  acids 
which  are  thus  set  free  combine  with  the  excess  of  alkali  to  form  soaps. 
The  process  of  the  hydrolysis  of  fats  by  alkali  is  therefore  known  as 
Saponification. 

Monoglycerides,  i.  e.,  glycerides  containing  only  one  fatty  acid 
molecule,  and  Diglycerides  are  readily  procurable  in  the  laboratory, 
but  they  do  not  usually  occur  in  natural  fats  unless  they  have  been 
exposed  to  the  action  of  fat-splitting  enzymes  (Lipases)  or  other  saponi- 
fying agencies.  In  the  Triglycerides  the  fatty-acid  radicals  need  not 
all  be  identical  and  two  or  even  three  different  fatty  acids  may  be 
combined  with  one  and  the  same  molecule  of  glycerol  to  form  neutral 
fat. 

The  specific  gravity  of  the  fats  is  less  than  that  of  water,  arid  when 
liquid,  or  liquefied  by  heat,  those  which  are  insoluble  in  water  float 
upon  the  top  of  it.  The  fats  which  are  formed  from  the  higher  fatty 
acids  are  insoluble  in  water,  while  the  solubility  of  the  lower  members 
in  water  decreases  as  the  number  of  carbon  atoms  in  the  fatty  acid 
molecule  increases.  They  are  soluble  in  a  variety  of  organic  solvents, 
and  form  very  stable  suspensions  or  emulsions  in  water  in  the  presence 
of  emulsifying  (surface-tension  reducing)  agents  such  as  soaps,  bile- 
salts,  saponins  and  so  forth. 

1  The  separation  of  glycerol  from  fats  was  first  accomplished  by  the  Swedish  chemist 
Scheele,  in  1779.  News  of  this  discovery  had,  however,  not  yet  reached  the  legislative 
assembly  of  one  of  the  allied  nations  in  1914,  with  the  result  that  in  1915  a  responsible 
official  of  the  executive,  in  reply  to  the  inquiry  of  a  legislator  stated  that  it  had  only 
recently  been  discovered  that  nitroglycerin  could  be  made  from  fats.  It  is  perhaps 
time  that  a  civilization  which  is  based  on  mechanics,  physics  and  chemistry  should  insist 
on  a  rudimentary  knowledge  of  the  practical  import  of  these  sciences  on  the  part  of  its 
legislators  and  executives. 


108  FATS 

The  fatty  acids  which  are  found  in  the  naturally  occurring  fats 
belong  to  two  series,  the  saturated  series,  represented  by  the  general 
formula  CnH2n02  and  the  unsaturated  or  oleic  acid  series  represented 
by  the  general  formula  CnH2n.2O2.  In  this  latter  series  of  acids  two 
of  the  carbon  atoms  are  united  by  a  double  bond  or  unsaturated  link- 
age which  enables  them  to  react  very  readily  with  hydrogen,  oxygen 
or  the  halogens,  the  double  bond  being  converted  into  a  single  one, 
and  the  remaining  valencies  of  the  carbons  saturated  by  combination 
with  the  reacting  atoms.  The  higher  fatty  acids  which  occur  in  nature 
usually  have  even  values  of  n  and  the  chain  of  carbon  atoms  is  not 
branched. 

The  lower  acids,  having  small  values  of  n,  are  formed  in  the  secre- 
tions of  the  sebaceous  glands,  and  in  butter,  while  the  tissue-fats  and 
vegetable  oils  are  in  the  main  composed  of  fats  derived  from  higher 
fatty  acids.  Thus  in  sweat  we  find: 

Formic  acid,  H.COOH 
Acetic  acid,  CH3COOH 
Propionic  acid,  C2H5COOH 
Butyric  acid,  CsHyCOOH 
Isovalerianic  acid,  C4H9COOH 
Caprylic  acid,  C7H15COOH 

These  acids  are  probably  secreted  in  combination  with  glycerol,  but 
if  the  sweat  be  allowed  to  remain  in  contact  with  the  skin,  the  glycerides 
are  attacked  by  bacteria  which  hydrolyze  them,  liberating  the  free 
acids,  to  which  the  characteristic  odor  of  the  "unwashed"  is  attribut- 
able. The  odor  .of  the  lowest  members  of  the  series,  Formic  and  Acetic 
Acids,  is  sharp,  reminiscent  in  the  former  instance  of  ants,  in  the  latter 
of  vinegar.  Propionic  Acid  has  an  intermediate  odor,  while  Butyric 
Acid  has  the  odor  of  rancid  butter  and  Valerianic  Acid  the  most  intensely 
disagreeable  odor  of  decomposing  perspiration.  The  highest  members 
of  the  fatty-acid  series  are  non-volatile  and  have  only  very  faint  odors. 

In  butter  the  lowest  member  of  the  series  is  butyric  acid,  while 
caproic  and  caprylic  acids  also  occur  together  with  higher  fatty  acids 
particularly  Palmitic  and  Oleic  Acids. 

The  majority  of  the  tissue -fats  are,  however,  mixtures  of  the  glycer- 
ides of  Palmitic  Acid,  Ci5H3iCOOH  and  Stearic  Acid  Ci7H35COOH,  of 
the  saturated  series,  with  glycerides  of  Oleic  Acid,  Ci7H33COOH,  of 
the  unsaturated  series.  The  separation  of  the  saturated  from  the 
unsaturated  acids  of  the  higher  series  may  be  accomplished  by  convert- 
ing them  after  hydrolysis  of  the  fat,  into  the  lead-salts,  and  extracting 
them  with  ether.  The  lead-salts  of  the  higher  fatty  acids  of  the  satur- 
ated series  are  almost  insoluble  in  ether,  while  those  of  the  unsaturated 
series  readily  dissolve  in  this  solvent.  The  ether  extract  therefore 
contains  all  of  the  unsaturated  fatty  acids  in  combination  with  lead. 
The  lead  may  be  removed  by  extraction  of  the  ether  with  dilute  hydro- 
chloric acid,  leaving  the  free  fatty  acids  dissolved  in  the  ether. 


CHARACTERISTICS  OF  THE  NATURAL  FATS  109 

THE  CHARACTERISTICS  OF  THE  NATURAL  FATS. 

The  various  animal  fats  and  vegetable  oils  differ  from  one  another 
very  strikingly  in  their  physical  characteristics  and  chemical  behavior. 
These  differences  are  in  the  main  determined  by  the  relative  propor- 
tions in  which  the  glycerides  of  the  three  fatty  acids  above  mentioned 
occur  in  the  fat.  The  glycerides  of  oleic  acid  have  the  lowest  melting- 
point,  those  of  stearic  acid  the  highest,  and  hence  olive'  oil,  which  con- 
sists very  largely  of  glycerol  trioleate  is  fluid  at  ordinary  tempera- 
tures, while  mutton-fat,  which  contains  a  high  proportion  of  glycerol 
tristearate,  is  solid  or  semi-solid  at  ordinary  temperatures.  The 
melting-points  of  the  pure  fats  are  as  follow: 

Triolein.      .      .      .      .      .     -.  •  ;     .    t.      .      .-    .     y     ....     -6.0°C. 

Tripalmitin 65.0°,C. 

Tristearin    ............'.'....'.      71. 5°  C. 

a  small  admixture  of  triolein,  however,  reduces  the  melting-point  of  a 
fat  to  a  very  considerable  degree. 

The  chemical  reactivity  of  the  fats  is  also  strongly  influenced  by 
their  content  of  oleates.  The  unsaturated  bond  in  oleic  acid  renders 
it  capable,  under  appropriate  conditions,  of  directly  absorbing  hydrogen, 
being  thereby  converted  into  the  corresponding  saturated  acid.  The 
artificial  hydrogenation  of  vegetable  oil  is  now  being  very  largely 
practised  and  results  in  the  production  of  a  solid  fat,  utilizable  for  a 
variety  of  household  purposes  for  which  the  fluid  oil  would  be  unsuited. 
The  significance  of  the  process,  however,  goes  far  beyond  this.  The 
addition  of  two  atoms  of  hydrogen  to  the  oleic  acid  molecule  adds 
considerably  to  its  calorific  value,  since  the  heat  of  combustion  of  the 
hydrogen  to  water  is  thus  rendered  available  for  nutritive  purposes. 
In  the  aggregate  the  hydrogenation  of  vegetable  oils  adds  to  the 
nutritive  value  of  these  fats  an  amount1  which  would  otherwise 
require  a  very  great  deal  of  space  and  labor  to  produce.  From 
an  economic  point  of  view  therefore,  and  as  a  means  of  food  con- 
servation, the  hydrogenation  of  vegetable  oils  is  a  very  desirable 
thing  to  encourage.  It  is  true  that  the  vegetable  oils  fail  in  important 
respects  to  furnish  the  nutritive  equivalent  of  animal  fats,  for,  as  we 
shall  see  in  later  chapters,  the  animal  fats  contain  accessory  foodstuffs 
which  are  essential  for  growth,  and  even  for  the  maintenance  of  health, 
while  the  vegetable  oils  are  lacking  in  these.  To  the  extent,  however, 
to  which  fats  are  employed  in  the  diet  for  their  mere  fuel-value,  the 
vegetable  oils  are  fully  equivalent  substitutes  for  the  animal  fats,  and 
only  a  small  proportion  of  the  total  fat-consumption,  at  any  rate  in 
adults,  is  requisite  to  furnish  the  accessory  foodstuffs  which  we  acquire 
from  the  animal  fats.  It  is  probable  that  there  would  be  no  danger  of 
shortage  of  the  accessory  foods  being  caused  by  the  utilization  of  vege- 

1  Roughly  7  per  cent. 


110  FATS 

table  fats,  unless  meat  and  dairy  products  were  at  the  same  time  very 
deficient  in  the  dietary. 

The  unsaturated  bonds  in  the  oleates  also  confer  upon  them  the 
property  of  absorbing  halogens,  and  the  power  of  various  natural 
fats  and  oils  to  absorb  iodine  is  used  as  a  means  of  characterizing  and 
identifying  them.  The  "Iodine  Number"  is  the  number  of  grammes  of 
iodine  which  is  absorbed  by  a  hundred  grammes  of  fat  dissolved  in 
chloroform  and  treated  with  a  solution  of  iodine  in  alcohol  or  acetic 
acid.  Other  characteristics  which  are  employed  to  differentiate  the 
natural  fats  are:  The  "Hehner  Number"  or  weight  of  water-insoluble 
fatty  acids  yielded  by  100  grammes  of  fat;  the  "Acid  Number"  or 
proportion  of  free  fatty  acid  in  the  fat,  estimated  by  titration  in 
alcoholic  solution;  the  "Reichert-Meissl  Number,"  or  proportion  of 
volatile  fatty  acids  yielded  by  distilling  the  hydrolyzed  fats  with 
steam;  the  Saponification-value  or  milligrammes  of  potassium  hydroxide 
neutralized  by  the  saponification  of  one  gramme  of  the  fat;  and 
the  Acetyl  Number,  or  amount  of  acetic  acid  yielded  by  1  gramme  of 
fat  after  treatment  with  hot  acetic  anhydride.  In  the  following  table 
the  melting-points,  iodine  numbers  and  saponification-values  of  some 
of  the  fats  most  commonly  employed  are  enumerated: 

Saponification 
Fat.  Melting-point.  Iodine  number.  value. 

Butter-fat 28°-33°  C.  26-  38  220-233 

Pork-fat 36°-46°  C.  50-  70  195-197 

Beef-fat       .      .      .      .      .  40°-48°  C.  36-48  193-200 

Sheep-tallow  44°-49°  C.  33-  46  192-195 


Human  fat 
Cod-liver  oil 
Cotton-seed  oil 
Olive  oil      .      . 
Linseed-oil 


17.5°  C.  57-  66  193-199 

0°-10°  C.  144-168  175-193 

3°-  4°  C.  105-117  191-196 

2°-10°  C.  78-  91  185-194 

-27°  C.  173-202  190-195 


Cod-liver  Oil  is  of  especial  interest  to  the  physician  because  of  its 
widespread  employment  as  a  food  and  therapeutic  agent  in  chronic  wast- 
ing diseases  such  as  tuberculosis,  and  rickets.  It  is  obtained  from  the 
livers  of  codfish  by  extraction  with  steam  and  water.  It  consists  of  a 
mixture  of  the  glycerides  of  a  great  variety  of  saturated  and  unsatur- 
ated fatty  acids  together  with  a  considerable  proportion  of  phos- 
pholipins,  a  small  amount  of  cholesterol,  numerous  nitrogenous  bases 
and  traces  of  iron,  manganese,  bromine  and  iodine.  The  therapeutic 
value  of  the  oil  has  been  variously  attributed  to  each  of  these  con- 
stituents in  turn,  and  on  the  other  hand  to  the  readily  digestible  char- 
acter  of  the  oil  itself.  Modern  opinion  inclines  to  the  view  that  the 
efficacy  of  cod-liver  oil  resides  mainly  in  its  high  calorific  value,  and 
the  fact  that  it  is  usually  added  in  considerable  dosage  to  the  pre- 
established  dietary.  On  the  other  hand  it  is  very  rich  in  accessory 
foodstuffs  and  the  possible  significance  of  some  of  these  must  not 
be  overlooked.  From  this  point  of  view  it  is  not  impossible  that  the 
therapeutic  applications  of  cod-liver  oil  may  be  destined  to  increase 
rather  than  to  diminish,  as  our  growing  knowledge  of  the  exact  require- 


WAXES  111 

ments  of  the  tissues  enables  us  to  use  it  with  more  judgment  and  less 
empirically  than  heretofore. 

Cotton-seed  Oil  consists  of  a  mixture  of  the  glycerides  of  oleic  and 
linoleic  and  palmitic  acids,  while  Olive  Oil  consists  almost  entirely 
(89  per  cent,  to  98  per  cent.)  of  the  triglyceride  of  oleic  acid. 

Linseed-oil  is  of  very  great  importance  in  the  industries  on  account 
of  its  peculiar  property  of  hardening  when  it  dries  in  thin  films  exposed 
to  the  air,  forming  a  transparent  waterproof  surface  and  accelerating 
the  drying  of  other  substances  (pigments,  etc.),  with  which  it  is  mixed. 
This  process  of  hardening  takes  place  at  first  slowly,  and  then  more 
rapidly,  the  products  of  oxidation  which  are  formed  accelerating  the 
further  stages  of  the  process.  The  oxidation  of  linseed-oil  which 
results  in  hardening  is,  in  fact,  an  "autocatalytic,"  that  is,  a  self- 
accelerated  reaction,  producing  its  own  catalyzers.  These  substances 
are  believed  to  be  unstable  peroxides  which  readily  break  down,  liber- 
ating oxygen  or  possibly  ozone,  which  oxidizes  adjacent  molecules  of 
the  oil.  Other  substances  which  accelerate  the  hardening  are  powdered 
lead,  zinc,  copper,  platinum  or  their  oxides.  This  phenomenon  depends 
upon  the  very  large  proportion  of  unsaturated  linkages  which  linseed 
oil  contains;  it  consists  of  a  mixture  of  the  glycerides  of  linoleic, 
linoleinic  and  isolinoleinic  acids  (fatty  acids  of  the  unsaturated  series 
Cnltn-eQj)  with  a  small  proportion  of  oleic,  palmitic  and  myristic 
acids,  and  a  trace  of  unsaponifiable  material. 

Castor-oil  is  obtained  by  expression  from  the  seeds  or  "beans"  of 
the  castor-oil  plant  (Ricinus  communis).  It  consists  in  the  main  of  the 
glycerides  of  ricinoleic  acid  Ci7H32(OH).COOH,  a  hydroxy-acid  of 
the  unsaturated  series.  It  is  without  aperient  action  until  saponified 
by  the  bile  and  pancreatic  juice  in  the  upper  part  of  the  sinall  intestine 
and  is  therefore  devoid  of  irritant  action  upon  the  walls  of  the  stomach. 

WAXES. 

In  addition  to  the  glycerides  of  fatty  acids  there  are  found  in  a 
variety  of  living  tissues  and  tissue-products,  fatty  acid  esters  of 
monatomic  alcohols  which  are  collectively  and  somewhat  loosely 
designated  Waxes.  This  term  is  not  infrequently  extended  to  include 
the  cholesterol  esters  of  the  fatty  acids,  for  no  better  reason  than  that 
cholesterol  is  a  monatomic  alcohol,  and  that  the  cholesterol  esters 
somewhat  resemble  the  waxes  in  certain  of  their  properties,  more 
particularly  in  the  difficulty  with  which  they  are  saponified.  It  would 
be  preferable,  however,  to  restrict  the  term  "wax,"  as  we  are  doing 
here,  to  the  fatty  acid  esters  of  the  higher  monatomic  alcohols  of  the 
paraffin  series.  In  this  way  we  will  include  in  the  class  all  of  the  most 
typical  waxes  of  commerce,  and  we  will  exclude  the  entirely  atypical 
esters  of  cholesterol. 

The  waxes  are  characterized  by  their  high  melting-point  and  the 
difficulty  with  which  they  are  saponified.  They  are  not  hydrolyzed 


112  FATS 

by  the  fat-splitting  ferments  (lipases),  and  it  is  only  with  comparative 
difficulty  that  they  are  split  into  their  components  .by  alkalies.  They 
are  hydrolyzed  by  bacteria,  and  hence  do  not  turn  sour  or  "rancid" 
on  standing.  While  their  high  melting-points  prevent  them  from  being 
"  sticky"  or  exhibiting  any  of  the  characteristic  properties  of  fluids  or 
oils  at  ordinary  temperatures,  yet  they  retain  the  "  greasy"  or  slippery 
qualities  of  the  fats  (more  accurately  expressed  as  the  possession  of  a 
low  coefficient  of  friction),  and  their  insolubility  in  water,  a  com- 
bination of  qualities  which  renders  them  ideal  and  indeed  indispens- 
able agents  for  polishing  and  waterproofing  the  surfaces  of  rough  or 
porous  materials. 

In  the  skull  of  the  white  whale  or  cachelot  (Physeter  macrocephalus) , 
there  is  found  a  large  cavity  which  during  life,  is  filled  with  an  oily 
liquid.  This  liquid  partially  solidifies  after  the  death  of  the  animal, 
and  consequent  fall  in  temperature,  and  separates  into  two  portions, 
a  solid  crystalline  part  ordinarily  called  Spermaceti  and  a  liquid  known 
as  Spermaceti-oil.  Spermaceti  is  also  found  in  some  other  whales 
and  in  certain  species  of  dolphins. 

Purified  spermaceti  is  a  mixture  of  fatty  acid  esters  of  monatomic 
alcohols.  The  chief  constituent  is  the  palmitic  acid  ester  of  Cetyl 
Alcohol,  CieH33OH,  mixed  with  small  quantities  of  the  lauric,  myristic 
and  stearic  acid  esters  of  the  twelve,  fourteen  and  eighteen  carbon 
atom  alcohols  of  the  paraffin  series  (general  formula  CnH2n+iOH). 

Spermaceti  is  used  for  making  "wax-candles,"  as  a  finishing  material 
or  waterproof  polish,  and  in  pharmacy  as  a  means  of  stiffening  emol- 
lients and  salves,  and  raising  their  melting-point,  particularly  in  hot 
climates.  Spermaceti-oil  is  a  very  valuable  lubricant  for  small  and 
delicate  machinery  or  apparatus. 

The  Beeswax  of  commerce  is  a  digestion-product  of  the  honey-bee, 
Apis  mellifica.  It  is  elaborated  by  special  glands  and  the  production 
of  honey  and  wax  stand  in  inverse  proportion  to  one  another,  the  pro- 
duction of  one  gramme  of  wax  diminishing  the  yield  of  honey  by  from 
ten  to  fourteen  grammes.  Regarding  the  mode  of  origin  of  the  wax 
from  the  foodstuffs  of  the  bee,  we  are  wholly  in  the  dark. 

The  chief  constituent  of  beeswax  is  the  palmitic  acid  ester  of  Myricyl 
Alcohol  C30H6iOH  with  an  admixture  of  other  acids  and  esters.  Bees- 
wax is  employed  in  a  variety  of  industries  too  numerous  to  mention 
here,  it  is  an  important  constituent  of  a  variety  of  commercial  waxes, 
which  are  prepared  by  the  admixture  of  paraffin  and  other  substances 
with  the  beeswax  to  obtain  the  combination  of  physical  qualities  which 
is  desired  for  the  purposes  for  which  the  wax  is  to  be  employed.  Adulter- 
ation with  paraffin  and  other  non-saponifiable  materials  may  be 
detected  by  the  low  saponification-value  of  the  mixture,  the  sapomfica- 
tion-value  of  pure  beeswax  lying  between  90  and  97. 

Waxes  are  produced  by  a  variety  of  insects,  notably  the  Hymenoi)- 
tera  (wasps  and  bees)  and  Homoptem  (cicadas  and  scale  insects). 
Japan  Wax  (or  Chinese  Wax)  is  obtained  from  a  scale-insect  which 


THE  PHOSPHOLIPINS  OR  PHOSPHATIDS  113 

infests  the  Chinese  Ash.  The  most  widely  employed  vegetable  wax 
is  carnaiiba  wax,  obtained  from  the  leaves  of  the  Wax-palm  or  Car- 
naiiba-palm  which  grows  in  tropical  South  America.  It  consists  of  a 
complex  mixture  of  esters  of  higher  monatomic  alcohols. 

THE  PHOSPHOLIPINS  OR  PHOSPHATIDS. 

In  all  living  tissues  and  without  exception,  we  find  a  variety  of 
complex  substances  resembling  the  fats  in  their  solubility  in  organic 
solvents,  and  yielding  fatty  acids,  alcohols  (usually  glycerol),  phos- 
phoric acid  and  nitrogenous  bases  when  hydrolyzed.  These  sub- 
stances constitute  the  group  of  Phospholipins,  and  on  account  of  their 
constant  association  in  the  tissues  with  cholesterol  and  cholesterol 
derivatives  they  are  sometimes  included  with  these  in  the  larger  group 
of  Lipoids  or  fat-resembling  substances,  the  common  characteristic 
of  the  group  consisting  in  their  high  solubility  in  fats  and  oils,  and  in 
the  various  fat-solvents.  The  term  lipoid,  however,  is  merely  a  con- 
venient brief  designation  of  a  heterogeneous  group  of  substances 
which  may  be  chemically  unrelated  to  one  another.  The  phospho- 
lipins,  on  the  contrary,  are  a  rather  well-defined  and  homogeneous 
group  of  chemically  related  substances. 

The  best  known  and  most  abundant  representatives  of  the  phos- 
pholipin  group  are  the  Lecithins.  These  substances,  which  are  found 
in  every  living  cell,  yield  fatty  acids,  glycerol,  phosphoric  acid  and 
choline  (=oxyethyl  trimethyl  ammonium  hydroxide)  on  hydrolysis. 
One  molecule  of  phosphoric  acid  is  yielded  for  every  molecule  of 
choline,  and  the  phosphorus  and  nitrogen-contents  of  these  substances 
stand  therefore  in  the  proportion  to  one  another  of  1  :  1.  The  struc- 
ture of  the  lecithins  is  believed  to  be  represented  by  the  formula: 

CHz.O — (fatty  acid  radical) 
CH.OL—  (fatty  acid  radical) 
CH2.0 
HO   -  P   =  O 

C2H4— O 

'  / 

N    =   (CH3)3 
\ 

OH. 

The  fatty  acid  radicals  consist,  as  a  rule,  of  palmitic,  stearic  or  oleic 
acid,  but  at  least  one  oleic  acid  radical  would  appear  to  be  invariably 
present,  since  the  lecithins  exhibit  to  a  very  high  degree  the  character- 

8 


114  FATS 

istic  instability  of  the  unsaturated  fatty  acids.  This  instability  is  in 
fact  enhanced  in  the  phospholipins  generally  to  a  remarkable  degree, 
and  the  difficulties  attending  their  preparation  and  purification  are 
rendered  exceptionally  great  by  their  extreme  susceptibility  to  oxi- 
dation. It  is  a  fact  which  is  doubtless  of  very  great  significance  that 
the  tissues  of  the  Brain  are  notably  rich  in  phospholipins,  while  the 
activities  of  the  brain  are  exceptionally  dependent  upon  an  abundant 
and  continuous  supply  of  oxygen,  the  first  bodily  activities  to  dis- 
appear in  asphyxia  being  those  of  the  higher  cerebral  centers. 

The  various  members  of  the  phospholipin  group  resemble  one 
another  very  closely  in  physical  and  chemical  behavior.  They  differ 
among  themselves  mostly  markedly,  first  in  the  proportion  of  phos- 
phorus to  nitrogen  which  they  contain,  and  secondly  in  their  solubilities 
in  certain  organic  solvents. 

Those  phospholipins  which,  like  lecithin,  contain  one  atom  of 
phosphorus  (i.  e.,  one  molecule  of  phosphoric  acid)  for  every  atom  of 
nitrogen,  are  termed  Monoamino-monophosphatids ;  those  which  con- 
tain two  molecules  of  phosphoric  acid  for  every  atom  of  nitrogen 
(P  :  N  =  2  : 1),  are  termed  Monoamino-diphosphatids ;  those  which 
contain  two  atoms  of  nitrogen  for  every  atom  of  phosphorus  are 
termed  Diamino-monophosphatids  (P  :  N  =  1  :  2),  and  so  forth. 
The  highest  proportion  of  nitrogen  to  phosphorus  which  has  been 
found  to  occur  in  a  phosphatid  is  that  of  four  atoms  of  nitrogen  for 
every  atom  of  phosphorus. 

The  majority  of  the  phospholipins  are  soluble  in  alcohol  and  in 
ether,  but  some  of  them  are  insoluble  in  ether,  and  others,  while 
soluble  in  alcohol  or  in  ether  alone,  are  insoluble,  or  but  sparingly 
soluble  in  certain  mixtures  of  the  two.  The  great  majority  of  the 
phospholipins,  but  not  all  of  them,  are  precipitated  from  ether  solu- 
tions by  the  addition  of  acetone,  a  fact  which  is  utilized  very  frequently 
in  their  preparation.  They  are  also  precipitated  by  a  variety  of 
metallic  salts,  and  platinum  chloride,  and  particularly  cadmium  chloride 
are  frequently  employed  for  their  separation  and  purification. 

The  phospholipins  are  amorphous  substances  which  are  generally 
white  or  cream-colored  when  pure,  but  darken  rapidly  on  exposure 
to  the  air.  The  iodine-number  simultaneously  diminishes,  indicating 
that  the  unsaturated  linkages  have  been  partially  neutralized  by  com- 
bination with  oxygen.  This  oxidation  is  particularly  accelerated  by 
heat  and  by  traces  of  moisture,  and  the  dried  or  partially  dried  phos- 
pholipins are  unfortunately  extremely  hygroscopic,  rapidly  attracting 
and  condensing  moisture  when  exposed  to  the  air.  The  drying  of 
phospholipins  without  decomposition  can  therefore  only  be  achieved 
at  low  temperatures,  and  in  vacuo  or  in  an  atmosphere  composed  of 
some  indifferent  gas.  There  seems  to  be  some  reason  for  supposing 
that  the  lability  of  the  phospholipins  may  be  greatly  enhanced  by 
impurities  which  are  commonly  associated  with  them,  and  that  in  the 
absence  of  these,  they  may  be  comparatively  stable. 


THE  PHOSPHOLIPINS  OR  PHOSPHATIDS  115 

The  majority  of  the  phospholipins  are  rapidly  hydrolyzed  by  the 
fat-splitting  ferments  or  Lipases,  yielding  fatty  acids,  glycero-phosphoric 
acid  and  nitrogenous  bases.  Glycero-phosphoric  acid  is  not  split  by 
lipase,  but  is  readily  decomposed  by  dilute  acids  yielding  phosphoric 
acid  and  glycerol.  Since  glycero-phosphoric  acid  is  not  liberated  from 
phospholipins  until  they  reach  the  small  intestine,  where  the  reaction 
is  alkaline,  it  would  appear  unlikely  that  it  is  split  before  absorption. 
According  to  some  authors,  an  enzyme  exists  in  tissue  extracts  from 
the  liver,  kidneys  and  intestinal  mucosa,  which  is  capable  of  bringing 
about  the  decomposition  of  glycero-phosphoric  acid,  but  the  constant 
presence  of  undecomposed  glycero-phosphoric  acid  in  small  amounts 
in  normal  urine  would  appear  to  render  this  doubtful.  Optically  in- 
active glycero-phosphoric  acid  is  readily  prepared  synthetically  from 
glycerol,  and  phosphoric  acid;  the  glycero-phosphoric  acid  yielded 
by  hydrolysis  of  phospholipins  is,  however,  levorotatory.  It  is  soluble 
in  water  and  insoluble  in  alcohol.  The  calcium  salt  is  readily  soluble 
in  cold,  but  almost  insoluble  in  boiling  water. 

The  Lecithins,  the  composition  and  probable  structure  of  which, 
have  already  been  discussed,  occur  in  all  plant  and  animal  cells.  A 
lecithin  or  a  mixture  of  lecithins,  is  particularly  abundant  in  the  yolks 
of  eggs,  and  may  be  obtained  in  impure  condition  by  extracting  the 
broken  yolks  with  ether,  and  adding  acetone  to  the  extract.  Lecithins 
are  particularly  abundant  in  young  and  rapidly  growing  or  embryonic 
tissues.  They  progressively  diminish  as  development  proceeds  and, 
in  the  embryos  of  sea-urchins  at  all  events,  they  are  probably  the 
source  from  which  the  phosphoric  acid  is  obtained  which  is  required  to 
build  up  the  nucleic  acids  in  the  nuclei  of  the  new  cells.  The  lecithins 
are  soluble  in  alcohol,  ether,  chloroform,  carbon  bisulphide,  benzol, 
and  fats  or  fatty  oils;  they  are  precipitated  from  ether  by  the  addition 
of  acetone.  In  water  they  swell  up  and  form  pasty  masses  which  throw 
out  oily  drops  and  threads,  the  so-called  "myelin  forms,"  into  the  body 
of  the  liquid.  This  probably  represents  the  beginning  of  an  imperfect 
emulsification,  and  the  addition  of  soaps,  saponins  or  bile-salts  acceler- 
ates and  completes  the  process  with  the  formation  of  relatively  stable 
milky  emulsions  which  are  coagulated  by  the  addition  of  small  quanti- 
ties of  salts  of  the  alkaline  earths  (calcium,  barium  or  strontium). 
The  lecithins  have  a  peculiar  greasy  odor  which  is  rather  sharp  and 
reminiscent  of  dried  brain-tissue.  They  are  tasteless. 

It  was  formerly  believed  that  lecithins  stood  in  a  peculiar  relation- 
ship to  certain  types  of  snake-venom  and  other  hemolyzing  poisons. 
If  a  pure  hemolyzing  snake-venom  be  allowed  to  act  upon  thoroughly 
washed  blood-corpuscles,  no  hemolysis  occurs.  Upon  the  addition  of 
blood-serum  or  of  impure  lecithin,  which  by  themselves  are  of  course 
without  action,  hemolysis  ensues  at  once.  It  has  been  ascertained 
by  Bang,  however,  that  pure  lecithin  prepared  from  yolk  of  eggs  is 
devoid  of  activating  influence  upon  cobra -venom.  It  would  thus 
appear  probable  that  the  activating  action  of  other  preparations  of 


116  FATS 

lecithin  is  attributable  to  impurities  which  may  nevertheless  be 
phospholipins.  Similarly  the  alleged  action  of  lecithin  in  "activating" 
fibrin-ferment  (the  blood-coagulating  ferment),  has  recently  been 
shown  to  be  due,  in  fact,  not  to  lecithin  but  to  kephalin. 

In  egg-yolk  and  in  a  variety  of  tissues  we  find  another  mono-amino- 
monophosphatid,  designated  Kephalin  which  differs  from  lecithin  in 
being  insoluble  in  alcohol.  It  is  particularly  abundant  in  brain-tissue 
and  may  be  extracted  by  dehydrating  the  tissue  with  acetone,  extract- 
ing with  ether  and  adding  alcohol  to  the  concentrated  extract.  It 
may  be  purified  by  precipitation  with  cadmium  chloride.  It  used 
to  be  alleged  that  kephalin  differed  from  lecithin  only  in  the  addition 
of  a  methyl  group,  but  recent  investigations  have  shown  the  difference 
to  be  much  more  profound,  inasmuch  as  the  nitrogenous  base  in  keph- 
alin is  not  cholin,  but  amino-ethyl  alcohol.  Besides  its  great  abun- 
dance in  brain-tissues,  where  it  doubtless  plays  an  important  role. 
Kephalin  is  of  very  great  physiological  importance  in  consequence  of 
the  part  which  it  plays  in  the  coagulation  of  the  blood.  The  recent 
investigations  of  Howell  and  McLean  have  shown  that  kephalin 
possesses  in  high  degree  the  power  of  neutralizing  the  anti-thrombin 
in  the  blood  and  thus  permitting  the  thrombin,  or  fibrin-ferment  to 
transform  fibrinogen  into  fibrin  and  bring  about  coagulation  of  the 
blood.  Very  small  amounts  of  kephalin,  therefore,  decisively  acceler- 
ate the  coagulation  of  the  blood  and  kephalin  is  now  being  utilized 
extensively  for  this  purpose  in  surgery. 

Cuorin  which  is  found  in  heart-muscle  is  a  mono-amino-diphosphatid 
(P  :  N  =  2  :  1).  It  is  insoluble  in  alcohol  and  in  acetone,  but  very 
soluble  in  ether,  chloroform  and  benzol.  It  readily  emulsifies  in  water 
and  on  the  addition  of  alkalies  the  emulsions  form  clear  solutions. 
The  nature  of  the  nitrogenous  base  in  cuorin  is  not  known.  It  con- 
tains three  fatty  acid  radicals  for  every  two  molecules  of  phosphoric  acid. 

Sphingomyelin,  which  is  found  in  the  brain,  is  a  diamino-monophos- 
phatid.  It  is  soluble  in  hot  alcohol  but  sparingly  soluble  in  cold 
alcohol,  and  insoluble  in  ether.  It  yields  on  hydrolysis  an  alcohol  of 
unknown  structure,  Sphingol  instead  of  glycerol,  and  it  yields'  two 
different  nitrogenous  bases,  namely  Neurine  and  a  base  of  which  the 
structure  is  not  yet  completely  defined,  which  is  designated  Sphingo- 
sine  (see  p.  197). 

GLUCOSIDES  OF  THE  PHOSPHOLIPINS. 

If  glucose  be  added  to  an  ether  solution  of  lecithin,  the  sugar,  which 
is  ordinarily  insoluble  in  ether,  dissolves  and  becomes  so  closely 
associated  with  the  lecithin  that  repeated  precipitation  and  resolution 
do  not  remove  it.  We  infer  that  lecithin  forms  a  compound  with 
glucose,  which,  however,  would  appear  to  be  a  very  loose  one,  since 
the  analyses  of  various  preparations  indicate  a  very  inconstant 
composition. 


GLUCOSIDES  OF  THE  PHOSPHOLIPINS  117 

In  the  tissues  of  the  liver  we  find  considerable  quantities  of  a  water- 
soluble  phospholipin,  Jecorin,  which  yields  glucose,  fatty  acids,  glycero- 
phosphoric  acid,  choline  and  hydrogen  sulphide  on  hydrolysis  by  alka- 
lies. Early  observers  were  inclined  to  regard  jecorin  simply  as  lecithin 
glucoside,  but  the  sulphur-content  of  jecorin  precludes  the  adoption 
of  this  simple  interpretation.  Jecorin,  like  other  phospholipins,  is 
exceedingly  hygroscopic  and  susceptible  to  oxidation.  It  is  soluble 
in  alcohol  containing  w^ater  and  in  ether,  but  is  insoluble  or  but  spar- 
ingly soluble  in  absolute  alcohol.  With  silver  nitrate  an  aqueous 
solution  of  jecorin  yields  a  precipitate  which  dissolves  in  excess  of  the 
jecorin  solution;  on  the  addition  of  ammonia  this  mixture  turns  dark 
red.  It  appears  highly  probable  from  the  variability  of  the  analytical 
data  obtained  with  jecorins  from  different  sources  that  a  variety  of 
substances  of  this  general  type  exist  in  the  tissues;  more  especially 
is  this  indicated  by  the  variable  ratio  of  phosphorus  to  nitrogen  which 
in  some  preparations  approximates  to  the  value  P  :  N  =  1  :  2,  while 
in  others  P  :  N  =  1  :  4.  In  any  case  it  is  probable  that  the  phospho- 
lipin portion  of  the  molecule  is  not  simply  lecithin.  All  preparations 
contain  sodium,  which  appears  to  be  present  in  chemical  combination. 

In  the  anterior  lobe  of  the  pituitary  gland  is  found  another  water- 
soluble  phospholipin  which,  however,  instead  of  yielding  glucose  on 
hydrolysis,  yields  the  cyclose  Inosite.  This  substance,  Tethelin,  is 
soluble  in  water,  alcohol  or  ether,  but  is  insoluble  in  a  mixture  of 
Alcohol  and  ether  in  the  proportion  of  one  part  by  volume  of  alcohol 
to  one  and  one-half  parts  by  volume  of  ether.  It  contains  phosphorus 
and  nitrogen  in  the  proportion  of  1  :  4  and  one-half  of  the  nitrogen 
is  present  in  the  molecule  in  the  form  of  amino-groups.  On  hydrolysis 
the  yield  of  amino-nitrogen  increases  to  three -fourths  of  the  total 
nitrogen,  indicating,  probably,  that  one  of  the  nitrogen  atoms  is 
present  in  the  form  of  an  imino-group  (=  NH). 

Tethelin  is  exceedingly  hygroscopic  and  susceptible  to  oxidation 
which  is  accelerated  by  traces  of  moisture.  Oxidation  is  accompanied 
by  progressive  darkening  of  the  originally  cream-colored  powder,  so 
that  it  ultimately  becomes  dark  brown  or  almost  black;  at  the  same 
time  the  iodine  absorption  number  decreases.  With  a  2  per  cent, 
solution  of  p-dimethylamino-benzaldehyde  in  hydrochloric  acid  cf 
sp.  gr.  1.09,  aqueous  solutions  of  tethelin  yield  a  pink  coloration 
(Ehrlich's  Reaction,  indicating  an  acetylated  oxyamino-acid  radical). 
The  nitrogenous  base  has  not  as  yet  been  identified,  but  it  probably 
contains  an  Iminazolyl  radical: 


This  is  of  great  significance  in  view  of  the  fact  that  the  active  prin- 
ciple of  the  Posterior  Lobe  of  the  Pituitary  Body  is  also  an  iminazolyl 
derivative  and  the  possibility  is  indicated,  'which  the  anatomical 


118  FATS 

relations  of  the  two  glands  tend  to  confirm,  that  the  active  principle 
of  the  posterior  lobe  is  derived  by  partial  decomposition  from  that  of 
the  anterior  lobe. 

Tethelin,  in  very  small  dosages  administered  by  mouth,  has  a 
remarkable  effect  upon  the  growth  of  animals,  consisting  in  the  main 
of  an  initial  retardation  followed  by  a  notable  acceleration  of  growth. 
These  phenomena  will  fall  under  fuller  consideration  in  a  later  chapter. 
When  administered  locally  in  aqueous  solution  or  incorporated  with 
lanoline  and  applied  as  a  salve  it  very  markedly  accelerates  the  repair 
of  slowly  healing  lesions  of  the  skin,  such  for  example  as  various 
ulcers.  The  effects  of  tethelin  upon  growth  almost  exactly  reproduce 
those  of  the  whole  anterior  lobe  tissue,  and  it  is  inferred  that  tethelin 
is  the  active  principle,  the  absence  or  superabundance  of  which  is 
responsible  for  the  remarkable  clinical  manifestations  of  hyperactivity 
(gigantism  and  acromegaly)  of  the  pituitary  gland. 

Tethelin  in  aqueous  solution  has  no  effect  upon  the  uterus,  and 
only  causes  in  very  large  dosages  a  slight  transient  fall  in  blood  pressure 
when  injected  intravenously  in  animals.  After  acidifying  the  solution 
(or  rendering  alkaline),  heating  for  a  brief  period  to  nearly  boiling- 
point  and  then  cooling  and  neutralizing,  however,  the  solution  has  a 
powerful  effect  in  causing  tonic  contractions  of  the  uterus  similar  to 
those  caused  by  extracts  of  the  posterior  lobe,  and  when  injected 
intravenously  causes  the  characteristic  rise  in  blood  pressure  which  is 
brought  about  by  small  doses  of  posterior  lobe  extract  (Pituitrin). 

In  the  tissues  of  the  brain  we  find  a  complex  substance  or  series 
of  substances  which  arise  by  combination  of  phospholipins  with  the 
cerebrosides  which  are  glucosides  yielding  galactose  on  hydrolysis. 
This  substance,  Protagon,  may  be  obtained  by  extracting  dehydrated 
brain-tissue  (dehydrated  by  acetone)  with  85  per  cent,  alcohol,  and 
cooling  to  zero  when  the  protagon  in  impure  form  is  precipitated.  On 
partial  hydrolysis  it  yields  sphingomyelin  (see  p.  116)  and  cerebrosides 
(see  p.  91). 

Protagon  when  dry  forms  a  fine  white  powder,  it  dissolves  in  85 
per  cent,  alcohol  at  45°  C.,  but  on  cooling  is  precipitated  in  groups  of 
small  acicular  crystals.  It  is  difficultly  soluble  in  cold  alcohol  or  ether, 
but  dissolves  in  warm  ether.  It  dissolves  in  methyl  alcohol  contain- 
ing chloroform  but,  on  standing,  this  solution  decomposes  and  a 
cerebroside,  Phrenosin  is  deposited.  In  water  protagon  swells  up  and 
forms  a  pasty  mass  which  dissolves  in  excess  of  water,  forming  an 
opalescent  solution.  Solutions  of  protagon  in  pyridin  are  dextro- 
rotatory, but  on  standing  they  decompose,  depositing  sphingomyelin, 
and  become  levorotatory. 

We  are  not  certain  whether  protagon  is  a  chemical  individual  or 
not.  The  composition  as  reported  by  different  observers,  varies 
very  considerably,  and  yet  preparations  have  been  obtained  which 
failed  to  alter  in  composition  or  optical  rotation  of  their  solutions  after 
repeated  resolution  and  recrystallization. 


GLUCOSIDES  OF  THE  PHOSPHOLIPINS  119 


REFERENCES. 

CHARACTERISTICS  OF  THE  NATURAL  FATS: 

Lewkowitsch:      Chemical    Technology    and    Analysis    of    Oils,    Fats    and    Waxes. 

London,  1913. 
THE  PHOSPHOLIPINS: 

Maclean:     Lecithin  and  Allied  Substances.     London,  1918. 

Levene  and  West:     Jour.  Biol.  Chem.,  1913-14,  16,  p.  419;  1916,  24,  pp.  41,  47,  50 
and  111;  1916,  25,  p.  517;  1918,  33,  p.  Ill;  1918,  34,  p.  175. 

McLean:     Am.  Jour.  Physiol.,  1916,  41,  p.  250;  1917,  43,  p.  586. 

Waksman:     Ibid.,   1918,  45,  p.   375. 
GLUCOSIDES  or  THE  PHOSPHOLIPINS  •, 

Rosenheim:     Biochem.  Jour.,  1914,  8,  p.  110;  1916,  10,  p.  142. 

Levene  and  West:     Jour.  Biol.  Chem.,  1917,  31,  p.  635. 

Koch  and  Koch:     Ibid.,   1917,  31,  p.  395. 

Robertson:     Ibid.,    1916,  24,  p.   409. 

Schmidt  and  May:     Jour,  of  Lab.  and  Clin.  Med.,  1916-1917,  2,  p.  708. 


CHAPTER  VII. 
THE  PROTEINS  AND  THE  AMINO-ACIDS. 

GENERAL  CHARACTERISTICS  OF  THE  PROTEINS. 

THE  greater  and  most  characteristic  part  of  the  organic  matter  in 
protoplasm  consists  of  colloidal  substances  containing  nitrogen  which 
are  designated  Proteins.  As  examples  of  the  proteins  we  may  recall 
white  of  egg,  which  is  practically  a  solution  of  protein  in  dilute  sodium 
chloride  solution,  or  casein,  which  is  flocculated  out  of  milk  by  the 
addition  of  acids,  and  gelatin,  which  is  derived  from  the  connective 
tissues  by  extraction  with  hot  water. 

The  proteins  all  contain  carbon,  hydrogen,  nitrogen  and  oxygen, 
while  the  great  majority  of  them  also  contain  sulphur  and  a  very 
great  many  of  them  contain  phosphorus.  Other  constituents,  for 
example,  iron,  copper  and  iodine  are  found  in  certain  exceptional 
proteins  or  in.  compounds  of  the  proteins  with  non-protein  radicals 
containing  these  elements.  The  average  composition  of  the  more 
typical  proteins  is  represented  in  the  following  table: 

Element.  Per  cent. 

C      .      .      . .      .     ....    .      .      .      .  50. 6  to  54.5 

H 6. 5  to    7.3 

N 15.0to  17.6 

S 0.3  to    2.2 

P 0.4to    0.9 

O 21. 5  to  23. 5 

When  perfectly  free  from  water,  the  proteins  form  loose  white 
powders,  but  when  imperfectly  dry,  and  especially  if  exposed  to  heat, 
they  tend  to  form  horny  semi-transparent  flakes  or  plates,  so  that  in 
most  of  the  older  literature,  before  the  modern  methods  of  dehydration 
at  low  temperature  by  absolute  alcohol  and  ether  were  employed, 
the  proteins  are  usually  described  as  horny  substances  when  in  the  dry 
condition. 

While  drying,  and  in  the  presence  of  traces  of  moisture  the  proteins 
show  a  marked  tendency  to  discoloration,  with  the  production  of 
heavily  pigmented  insoluble  substances  which  are  probably  related 
to  the  "  humin-substances"  which  are  produced  in  the  presence  of 
carbohydrates  by  boiling  the  tryptophane  radical  of  proteins  with 
acids.  Many  proteins  have  curious  and  characteristic  faint  odors, 
but  they  are  generally  tasteless  and  amorphous. 

Notwithstanding  their  colloidal  character  and  very  slight  diffusi- 
bility  in  solutions,  many  proteins  may,  nevertheless,  under  suitable 


GENERAL  CHARACTERISTICS  OF  THE  PROTEINS          121 

conditions,  be  obtained  in  crystalline  condition.  This  is  particularly 
true  of  hemoglobin,  of  egg-albumin,  the  serum-albumin  of  the  horse, 
and  a  variety  of  vegetable  proteins.  The  solutions  of  the  proteins 
are  always  optically  active  and  with  the  exception  of  the  solutions  of 
hemoglobin  and  the  nucleo-proteins,  are  levorotatory. 

The  great  majority  of  the  proteins  are  soluble  in  water  or  in  very 
dilute  acids  or  alkalies.  Some  exceptional  proteins,  however,  such  as 
Elastin  from  elastic  fibers  of  connective  tissues,  Keratin  from  horn, 
Fibroin  from  silk  and  Spongin  from  sponges,  are  insoluble  in  water  or 
in  dilute  acids  and  alkalies  and  require  strong  acids  or  alkalies  to  bring 
them  into  solution.  The  action  of  strong  soda  upon  a  sponge  may 
be  cited  in  illustration.  The  proteins  are  usually  insoluble  in  organic 
reagents,  although  some  of  the  vegetable  proteins,  particularly  those 
obtained  from  a  variety  of  grains,  are  soluble  in  80  per  cent,  alcohol. 
Many  of  the  proteins  not  commonly  regarded  as  alcohol-soluble  are, 
however,  soluble  in  faintly  alkaline  alcohol,  if  they  are  first  dissolved 
in  alkaline  water,  and  alcohol  added  up  to  80  or  90  per  cent.  Casein 
is  soluble  in  warm  anhydrous  formic  acid,  but  the  protein  undergoes 
decomposition  if  the  solution  is  allowed  to  stand. 

The  proteins  combine  with  both  acids  and  bases,  neutralizing  them 
wholly  or  in  part,  and  causing  a  diminution  of  hydrogen  ions  in  the  case 
of  combination  with  acids,  or  of  hydroxyl  ions  in  the  case  of  combi- 
nation with  bases.  They  therefore  belong  to  the  class  of  substances 
designated  Amphoteric  Acids,  or  acids  which  are  simultaneously 
capable  of  acting  as  bases.  Under  certain  conditions  the  proteins 
are  also  capable  of  combining  with  neutral  salts. 

When  dissolved  in  water,  especially  in  faintly  acid  solutions,  the 
proteins  are  usually  modified  by  heat  in  such  a  way  as  to  render  them 
less  soluble.  This  generally  leads  to  flocculation  or  Coagulation  of  the 
protein,  or  if  the  solution  be  very  concentrated,  to  the  formation  of  a 
firm  jelly,  such  as,  for  example,  the  white  of  a  hard-boiled  egg. 

The  true  characterization  of  the  proteins  depends  upon  the  presence 
among  their  hydrolytic  cleavage  products  of  a  preponderating  pro- 
portion of  Amino-acids.  No  other  single  "test"  can  be  relied  upon 
to  demonstrate  the  presence  of  a  protein  in  a  solution  containing 
unknown  substances,  nor  can  the  individual  proteins  be  accurately 
characterized,  as  a  general  rule,  in  any  other  terms  than  the  propor- 
tions of  various  cleavage-products  which  they  yield  on  hydrolysis. 
By  employing  a  multiplicity  of  tests,  however,  the  presence  of  protein 
in  a  solution  may  be  established  by  the  fact  that  the  unknown  sub- 
stance yields  several  positive  reactions.  For  the  identification  of  any 
particular  protein  we  depend  upon  slight  peculiarities  of  solubility 
in  various  salt  solutions,  dilute  acids  and  alkalies,  etc.,  and  upon 
physical  peculiarities  and  the  nature  of  the  tissue  or  fluid  in  which  the 
protein  occurs. 

The  various  reactions  which  the  majority  of  the  proteins  yield  may 
be  subdivided  into  Coagulation-reactions  which  involve  or  depend 


122  THE  PROTEINS  AND  THE  AMINO-ACIDS 

upon  dehydration  of  the  protein  and  the  formation  of  complex  insoluble 
anhydrides,  Precipitation-reactions,  which  depend  upon  the  formation 
of  insoluble  compounds  with  the  precipitating-agents  employed,  and 
Color-reactions  which  depend  upon  chemical  interaction  with  the 
reagents  employed,  resulting  in  the  production  of  distinctive  colors. 
The  most  important  of  these  reactions  are  the  following: 

COAGULATION-REACTIONS. 

1.  Heat. — Heat  applied  to  solutions  acidified  with  ace_tic  acid.     If 
mineral  acids  are  employed,  compounds  of  the  protein  with  the  acid 
may  be  formed  which  are  incoagulable  by  heat. 

2.  Alcohol. — Alcohol  added  to  neutral  or  acid  solutions. 

3.  Concentrated  Neutral  Salts. — Concentrated  neutral  salts,  particu- 
larly ammonium  sulphate  or  magnesium  sulphate.  In  ajcidified  solutions 
concentrated  sodium  chloride  or  sodium  sulphate  are  also  coagulants  of 
protein. 

4.  Strong  Mineral  Acids. — Upon  the  ability  of  the  strong_mineral 
acids  to  coagulate  proteins  depends  Heller's  Test  for  protein  in  urine. 
The  suspected  sample  of  urine  is  placed  in  a  test  tube  and  concen- 
trated nitric  acid  is  allowed  to  flow  into  the  bottom  of  the  tube  from 
a  pipette.    At  the  junction  of  the  two  fluids  a  white  ring  of  coagulated 
protein  is  formed. 

Precipitation  Reactions. — 1.  The  Salts  of  Heavy  Metals,  such  as 
cupric  sulphate,  lead  acetate,  mercuric  chloride,  silver  nitrate,  etc., 
form  insoluble  compounds  with  proteins.  In  the  presence  of  excess 
of  the  reagent,  the  precipitate  which  at  first  forms  not  infrequently 
redissolves. 

.  2.  The  so-called  Alkaloidal  Reagents,  such  as  phosphotungstic  or 
phosphomolybdic  acids,  tannic  acid,  potassium  mercuric  iodide,  picric 
acid,  trichloracetic  acid,  phenol  and  salicyl-sulphonic  acid.  Other 
reagents  which  similarly  precipitate  proteins  are  metaphosphoric  acid, 
nucleic  acids,  chondroitin-sulphuric  acid  and  taurocholic  acid.  Potassium 
ferrocyanide  and  acetic  acid  yield  an  insoluble  compound  of  the  protein 
with  hydroferrocyanic  acid. 

Color -reactions. — 1.  Millon's  Reaction. — A  solution  of  mercury  in 
strong  nitric  acid  yields  a  mixture  of  mercuric  and  mercurous  nitrates 
dissolved  in  a  mixture  of  nitric  and  nitrous  acids.  If  this  reagent 
be  added  to  a  protein  solution,  a  precipitate  is  produced  which  turns 
brick-red  on  heating. 

2.  The  Xanthoproteic  Reaction. — On   adding   strong   nitric   acid   to 
protein  solutions  and  heating  to  boiling,  a  pale  yellow  solution  or 
coagulum  results.    On  rendering  the  mixture  alkaline  with  ammonia 
it  becomes  orange-yellow. 

3.  The  Hopkins-Cole  Reaction. — A  solution  of  glyoxylic  acid,  formed 
by  acting  upon  a  concentrated  solution  of  oxalic  acid  with  sodium 
amalgam,  is  added  to  the  protein  solution  in  a  test  tube,  and  sulphuric 


COAGULATION-REACTIONS  123 

acid  introduced  at  the  bottom  of  the  tube  by  means  of  a  pipette.     A 
reddish- violet  ring  is  formed  at  the  junction  of  the  two  liquids. 

4.  Acree's  Reaction. — To  the  solution  is  added  an  equal  volume 
of  a  0.02  per  cent,  solution  of  formaldehyde  containing  a  trace  of 
ferric  chloride.    Concentrated  sulphuric  acid  is  then  introduced  below 
the  mixture  and  at  the  junction  of  the  two  fluids  a  violet  ring  is  formed. 

5.  The  Biuret-reaction. — The  protein  solution  is  rendered  strongly 
alkaline  with  concentrated  sodium   or  potassium  hydroxide,  and  j, 
dilute  solution  of  cupria^ulphate  ls_added,  one^drop  at  a  time.     A 
reddish  or  bluish  violet  results  in  solutions  of  proteins,  and  a  pink 
color  in  solutions  of  their  digestion-products,  the  peptones.     Excep- 
tions are  afforded  by  the  protamiiie  group  of  proteins,  which  yield  a 
pink  biuret-reaction  without  preliminary  hydrolysis. 

6.  The  Ninhydrin  Reaction.— One-tenth  of  a  gram  of  Triketohydrin- 

denehydrate  ("Ninhydrin"): 

CO 


is  dissolved  in  from  thirty  to  forty  c.c.  of  water,  one  or  tw.o  drops 
of  this  solution  are  added  to  one  c.c.  of  the  protein  solution,  and  the 
mixture  is  heated  for  a  short  time  to  boiling.  On  cooling,  an  intense 
blue  or  bluish  violet  color  develops.  This  reaction  is  given  not  only 
by  proteins,  but  also  by  their  cleavage-products,  proteoses,  peptones 
and  even  amino-acids,  with  the  exception  of  proline  and  oxyproline. 
This  reaction  is  exceedingly  delicate  and  is  given  by  substances  con- 
taining at  least  one  free  carboxyl-group,  and  one  amino-group;  it  will 
detect  glycine  (amino-acetic  acid)  in  solutions  containing  only  one  part 
in  ten  thousand.  This  extreme  delicacy,  in  fact,  renders  the  reaction 
rather  unserviceable  as  a  practical  test  for  proteins  or  their  decom- 
position-products, since  such  extraordinary  precautions  have  to  be 
taken  to  ensure  that  a  positive  test  may  not  have  been  attributable  to 
accidental  contamination  of  reagents  or  apparatus  with  traces  of  the 
many  substances  that  will  yield  a  positive  reaction. 

All  of  these  color  reactions,  with  the  exception  of  the  biuret  reaction 
and  the  ninhydrin  reaction,  depend"  upon  specific  atomic  groupings 
which  are  usually,  but  not  invariably  present  in  the  protein  molecule. 
Thus,  Millon's  reaction  is  attributable  to  a  hydroxy-benzene  radical 
which  is  usually  present  in  proteins  in  the  form  of  the  amino-acid 
Tyrosine,  but  is  absent  from  certain  proteins,  for  example  gelatin.  The 
xanthoproteic  reaction  is  attributable  to  aromatic  groups  which  are 
provided  by  the  Tyrosine,  Phenylalanine  and  Tryptophane  radicals  in  the 
protein  molecule.  The  xanthoproteic  reaction  is  therefore  not  given 
by  proteins  such  as  the  members  of  the  protamine  group  in  which 


124  THE  PROTEINS  AND  THE  AMINO-ACIDS 

these  radicals  are  lacking.    The  Hopkins-Cole  reaction  is  attributable 

to  the  indole  linkage: 

c— 


C6H4  CH 

\  / 


NH 

which  is  present  in  the  tryptophane  (indole  aminopropionic  acid) 
radical  of  protein.  It  is  therefore  not  given  by  proteins  from  which 
this  radical  is  absent,  such  as,  for  example,  zein,  a  protein  obtained 
from  corn  (maize). 

The  biuret-reaction,  on  the  contrary,  is  yielded  by  a  variety  of  sub- 
stances such  as  oxamide,  biuret,  etc.,  which  are  not  proteins.  The 
Deaminized  Proteins  which  are  laboratory-products  formed  by  acting 
upon  proteins  with  nitrous  acid,  and  in  which  the  NH2  groups  have 
been  replaced  by  hydroxyl-groups,  no  longer  give  the  biuret-reaction, 
although  they  otherwise  resemble  the  natural  proteins  in  physical  and 
chemical  behavior. 

THE  CLASSIFICATION  OF  THE  PROTEINS. 

American  and  English  biochemists  have  unfortunately  adopted 
slightly  different  systems  of  classification  of  the  proteins.    The  Ameri- 
can classification,   adopted  by  the  American  Physiological  Society 
and  the  American  Society  of  Biological  Chemists,  is  as  follows: 
I.  SIMPLE  PROTEINS: 
Albumins. 
Globulins. 
Glutelins. 

Prolamins  (alcohol-soluble  proteins). 
Albuminoids. 
Histones. 
Prot  amines. 

H.  CONJUGATED  PROTEINS: 
Nucleoproteins. 
Glycoproteins. 
Phosphoproteins. 
Hemoglobins. 
Lecithoproteins. 
HI.  DERIVED  PROTEINS: 

1.  Primary  Protein  Derivatives: 

Proteans. 
Met  a  proteins. 
Coagulated  Proteins. 

2.  Secondary  Protein  Derivatives: 

Proteoses. 
Peptones. 
Peptides. 


THE  CLASSIFICATION  OF  THE  PROTEINS  125 

The  classification  adopted  by  the  British  Medical  Association  is  the 
following : 

I.  SIMPLE  PROTEINS: 

Protamines. 

Histones. 

Albumins. 

Globulins. 

Glutelins. 

Alcohol-soluble  Proteins. 

Scleroproteins.  +- 

Phosphoproteins.  V 
H.  CONJUGATED  PROTEINS: 

Glucoproteinsr 

NucleoproteinsM 

Chromoproteins.  * 
m.  PRODUCTS  OF  PROTEIN  HYDROLYSIS: 

Infraproteins. 

Proteoses. 

Peptones. 

Polypeptides. 

. 

Neither  of  these  systems  of  classification  is  free  from  objection. 
To  them  both  the  general  objection  applies,  that  the  distinctions 
drawn  are  largely  based  upon  variations  in  physical  behavior  which 
do  not  necessarily  correspond  to  fundamental  differences  of  chemical 
architecture  while,  on  the  other  hand,  many  protein  or  protein-like 
substances  are  known  which  display  intermediate  characteristics,  or 
individual  peculiarities  which  render  their  inclusion  in  any  of  the  classes 
enumerated,  a  matter  of  more  or  less  arbitrary  opinion.  In  particular 
the  defect  of  the  American  system  lies  in  the  rather  intangible  dis- 
tinctions which  are  made  between  various  classes  of  primary  protein 
derivatives,  and  the  inclusion  of  coagulated  proteins  which  are  almost 
certainly  derived  from  native  proteins  by  abstraction  of  water  from 
the  molecule,  in  the  same  class  with  proteans  and  metaproteins  which 
are  derived  from  native  proteins  by  partial  hydrolysis,  is  unfortunate. 
The  English  classification  has  the  merit  of  simplicity,  but  it  would  be 
more  advisable  to  include  the  phosphoproteins  among  the  conjugated 
proteins,  as  in  the  American  classification,  and  to  add  a  fourth  group 
to  accommodate  the  coagulated  proteins.  The  term  prolamine  is  also 
preferable  to  the  designation  a  alcohol-soluble  proteins,"  because  it 
draws  attention  to  the  high  content  of  the  amino-acid  proline  which 
characterizes  these  proteins  more  fundamentally  than  the  physical 
property  of  solubility  in  eighty  per  cent,  alcohol. 

The  salient  characteristics  of  these  various  classes  of  protein  sub- 
stances are  as  follows: 


126  THE  PROTEINS  AND  THE  AMINO-ACIDS 

I.  THE  SIMPLE  PROTEINS. 

Protamines. — The  protamines  are  the  simplest  proteins  which  are 
as  yet  definitely  known  to  occur  in  nature.  They  are  found  in  sperma- 
tozoa, and  especially  in  the  spermatozoa  of  fishes  in  combination  with 
nucleic  acid,  forming  a  simple  type  of  nucleoprotein.  They  are  pre- 
dominantly basic  substances,  indeed  so  strongly  basic  that  a  solution 
of  salmine  (the  protamine  from  salmon  spermatozoa)  reacts  alkaline 
to  litmus  and  absorbs  carbon  dioxide  from  the  air,  forming  carbonates 
of  the  protamine.  The  acid  function  of  these  substances  is  correspond- 
ingly weak,  although  they  are,  like  all  proteins,  amphoteric  acids,  and 
in  the  presence  of  excess  of  strong  bases  will  partially  combine  with 
them. 

The  protamines  are  soluble  in  water  and  form  definite  salts  with 
acids  which  are  coagulated  by  alcohol  and  thrown  out  of  solution 
without  decomposition,  the  combined  acid  being  carried  down  quanti- 
tatively with  the  protein.  They  yield  a  pink  biuret-reaction  resembling 
in  this  respect  the  derivatives  of  the  partial  hydrolysis  of  other  native 
proteins.  They  yield,  when  completely  hydrolyzed,  a  preponderating 
proportion  of  diamino-acids. 

Histones. — The  histones  are  somewhat  more  complex  and  colloidal  in 
character  than  the  protamines,  and  their  basic  function  is  less  marked. 
They  are  still  predominantly  basic,  however,  and  occur,  in  cellular 
tissues,  combined  with  nucleic  acid,  and  in  the  chromoprotein,  hemo- 
globin, combined  with  a  colored  acid  radical,  Hematin.  They  are 
soluble  in  dilute  acids  or  dilute  solutions  of  the  strong  bases,  but  are 
precipitated  from  acid  solutions  by  the  addition  of  ammonia. 

Albumins. — The  albumins  are  markedly  colloidal  substances  which 
are  soluble  in  distilled  water  and  in  salt  solutions.  The  basic  function 
is  almost  equal  to  the  acid  function.  Representative  examples  are  egg- 
albumin  and  the  albumin  which  is  found  in  blood-serum.  They  are 
coagulated  by  saturation  of  their  solutions  with  ammonium  sulphate. 

Globulins. — The  globulins  are  very  decidedly  colloidal  substances 
passing,  for  example,  with  difficulty,  or  not  at  all  through  clay  filters. 
They  are  insoluble  in  distilled  water,  but  are  soluble  in  dilute  solutions 
of  strong  acids  or  bases,  or  of  inorganic  salts.  The  acid  function  pre- 
dominates slightly  over  the  basic,  so  that  they  neutralize  bases  more 
readily  and  completely  than  acids.  Typical  examples  are  afforded  by 
serum-globulin,  the  globulin  which  is  precipitated  from  egg-white 
by  dilution  with  distilled  water,  and  a  variety  of  vegetable  proteins 
such  as  edestin,  obtained  from  seeds -of  hemp  (Cannabis  Sativa).  They 
are  coagulated  by  half-saturation  of  their  solutions  with  ammonium 
sulphate  or  complete  saturation  with  magnesium  sulphate. 

Glutelins. — The  glutelins  are  a  group  of  vegetable  proteins  of  which 
only  two,  the  Glutenin  of  wheat  and  the  Oryzenin  of  rice  have  as  yet 
been  prepared  in  sufficient  quantity,  and  purity  to  render  analysis 
and  characterization  possible.  They  are  insoluble  in  water  or  dilute 


THE  SIMPLE  PROTEINS  127 

salt  solutions  but  they  are  soluble  in  dilute  solutions  of  strong  bases 
or  acids. 

Prolamins. — The  prolamins  are  soluble  in  70  per  cent,  to  90  per  cent, 
alcohol.  They  are  insoluble,  or  nearly  so,  in  distilled  water,  but  dis- 
solve readily  in  dilute  solutions  of  strong  acids  or  bases.  They  occur 
in  a  variety  of  grains,  typical  members  of  the  group  being  Gliadin, 
found  in  the  seeds  of  wheat  and  rye,  Hordein  found  in  the  seeds  of 
barley  and  Zein  found  in  the  seeds  of  maize.  They  are  characterized 
by  the  high  proportion  of  Proline  which  they  yield  when  hydrolyzed. 

Scleroproteins. — The  scleroproteins,  termed  albuminoids  in  Ameri- 
can and  Continental  European  publications,  form  a  very  heterogeneous 
group  of  substances.  The  various  proteins  which  we  have  hitherto 
been  considering  are  either  constituents  of  cellular  tissues,  concerned 
in  the  life  and  maintenance  of  the  protoplasm,  or  else  they  form 
reserve-materials  which  are  sooner  or  later  to  be  called  upon  to  supply 
the  requirements  of  protoplasm.  Quite  other  is  the  function  of  the 
scleroproteins,  for  these  are  proteins  of  a  primarily  structural  or 
architectural  rather  than  nutritional  significance.  They  are  binding, 
cementing  and  supporting  substances  which  contribute  in  a  mechanical 
rather  than  in  a  chemical  fashion  to  the  furtherance  and  maintenance 
of  life.  They  occur  especially  in  the  various  connective  tissues,  and 
corresponding  with  their  peculiar  function,  we  find  that  they  display 
a  variety  of  physical  characteristics,  distinguishing  them  from  the 
proteins  of  cellular  origin,  and  also  distinguishing  the  individual 
members  of  the  group  very  sharply  from  one  another.  Typical 
members  of  this  class  are  Gelatin  and  its  parent-substance  Collagen 
which  forms  the  chief  constituent  of  white  fibrous  connective  tissue, 
and  also  the  main  organic  constituent  of  bones.  On  boiling,  especially 
in  the  presence  of  dilute  acid,  Collagen  yields  the  cleavage-product 
Gelatin.  Collagen  itself  is  insoluble  in  water,  salt  solutions  and  dilute 
acids  or  alkalies,  but  gelatin  swells  in  cold  water  and  dissolves  in 
warm  water,  forming  jellies  on  cooling  if  the  solutions  are  sufficiently 
concentrated.  Reticulin,  occurring  in  the  reticular  fibrous  tissues  of 
glands  differs  from  collagen  in  several  respects,  notably  in  containing 
phosphorus. 

Keratin  is  another  scleroprotein  and  forms  the  chief  constituent 
of  the  horny  epidermal  structures,  hair,  wool,  nails,  hoofs,  horns, 
feather,  tortoise-shell,  etc.  A  form  of  keratin,  Neurokeratin,  also 
occurs  in  nervous  tissues.  Keratin  is  insoluble  in  water,  dilute  acids  or 
alkalies  and  salt  solutions;  it  is  soluble  with  difficulty  in  strongly 
alkaline  solutions.  It  is  also  characterized  by  the  high  percentage  of 
sulphur  wrhich  it  contains  and  which  is  attributable  to  the  amino-acid 
radical  Cystine. 

Elastin  forms  the  chief  constituent  of  the  elastic  fibers  of  connective 
tissue.  It  is  distinguished  by  its  elasticity  and  tensile  strength  and 
also  by  its  extreme  insolubility,  being  soluble  only  in  strong  caustic 
alkalies  or  concentrated  mineral  acids.  Fibroin,  the  substance  forming 


128  THE  PROTEINS  AND  THE  AMINO-ACIDS 

the  core  of  silk  fibers,  is  characterized  by  possibly  even  greater  tensile 
strength,  while  it  is  somewhat  more  readily  dissolved  by  concentrated 
acids  and  alkalies  than  elastin.  Sericin  or  silk  gelatin  forms  the  outer 
coating  of  the  silk  fiber,  and  is  sticky  when  freshly  secreted,  so  that  it 
enables  intersecting  and  adjacent  fibers  to  adhere.  It  is  soluble  in  hot 
water,  and  the  solution  resembles  a  solution  of  ordinary  gelatin  in 
that,  if  concentrated,  it  gelatinizes  on  cooling.  Finally,  Spongin  forms 
the  chief  part  of  the  ordinary  sponge  from  which  the  originally  living 
protoplasm  has  been  extracted.  It  is  insoluble  in  acids  but  soluble  in 
concentrated  alkalies.  Some  of  the  spongins  contain  iodine  as  an 
integral  part  of  the  molecule. 

The  scleroproteins  are  for  the  most  part  incomplete  proteins  in  the 
sense  that  they  do  not  yield  when  completely  hydrolyzed,  all  of  the 
amino-acids  that  we  are  accustomed  to  obtain  from  the  more  typical 
proteins  of  cellular  tissues.  Thus  gelatin  yields  neither  tyrosine  nor 
trypto;phane,  elastin  and  fibroin  yield  neither  aspartic  nor  glutamic 
acids,  and  spongin  yields  neither  tyrosine  nor  phenylalanine. 

The  extraordinary  variety  of  physical  properties  and  peculiarities 
displayed  by  the  various  scleroproteins  reveals  the  possibility  of  sub- 
stances of  very  unique  physical  characteristics  being  derived  from 
proteins,  and  would  point  to  the  ultimate  possibility  of  very  important 
industrial  applications  of  such  derivatives.  At  the  present  time,  horny 
derivatives  of  the  protein  of  milk,  casein,  are  extensively  used  in  the 
manufacture  of  substitutes  for  ivory,  celluloid  and  bone.  The  animal 
proteins,  being  among  the  most  expensive  foodstuffs  we  require,  can 
never  be  employed  very  extensively  in  the  industries,  except  ing  when 
they  form  by-products  of  the  foodstuffs-industry,  as  in  the  manufacture 
of  glue  from  slaughter-house  or  fish-  wastes,  and  of  casein  products  from 
skimmed  milk.  Certain  vegetable  proteins  might,  however,  be  rendered 
relatively  cheap  and  abundant  and  offer  an  interesting  field  for  the 
investigation  of  the  special  physical  characteristics  of  their  derivatives. 

H.  THE  CONJUGATED  PROTEINS. 

Nucleoproteins. — The  conjugated  proteins  are  complex  substances 
formed  by  the  union  of  a  protein  with  a  non-protein  radical,  which 
may  be  termed  the  Prosthetic  Group.  The  Nucleoproteins,  for  example, 
are  compounds  of  Nucleic  Acids,  which  are  substituted  phosphoric 
acids  containing  carbohydrate  and  nitrogenous  radicals,  with  a  protein 
which  plays  the  part  of  a  base  in  the  compound.  These  compounds 
are  the  most  characteristic  constituents  of  the  nuclei  of  cells.  When 
the  protein  constituent  is  a  histone,  the  compound  is  termed  a  Nucleo- 
histone. 

The  nucleoproteins  are  insoluble  in  distilled  water,  but  soluble  in 
dilute  alkalies  from  which  solutions  they  are  precipitated  by  weak 
acids,  such  as  acetic  acid  or  carbon  dioxide.  They  are  as  a  rule  incom- 
pletely digestible  by  the  pepsin  of  gastric  juice,  leaving  an  indigestible 


THE  CONJUGATED  PROTEINS  129 

residue  which  still  contains  protein  and  is  termed  Nuclein.  All  of  the 
nucleoproteins  appear  to  be  very  closely  associated,  or  possibly  com- 
bined with  Iron. 

Glucoproteins. — In  the  glucoproteins  the  prosthetic  group  is  either, 
an  ammo-carbohydrate,  a  polysaccharide  derived  from  glucosamin 
or  acetylated  derivatives  of  glucosamin,  or  else  chondroitin-sulphuric 
acid  (see  p.  91).  The  glucoproteins  are  subdivided  into  Mucins, 
Mucoids  and  Chondroproteins.  The  true  mucins  yield  extraordinarily 
glutinous  or  mucilaginous  solutions  from  which  the  mucin  is  precipi- 
tated by  acetic  acid.  The  mucoids  are  not  precipi table  by  acetic  acid 
and  do  not,  as  a  rule,  yield  such  highly  viscous  solutions  as  the  mucins. 
The  chondroproteins  are  insoluble  in  water,  but  are  soluble  in  dilute 
alkalies,  from  which  solutions  the  protein  is  precipitated  by  neutral- 
ization with  strong  acids  or  by  an  excess  of  acetic  acid.  They  yield 
Chondroitin-sulphuric  Acid  on  hydrolysis,  a  substituted  sulphuric  acid 
formed  by  the  union  of  a  molecule  of  Chondroitin  with  a  molecule  of 
sulphuric  acid.  Chondroitin  resembles  gum-arabic  in  physical  char- 
acteristics, and  is  a  compound  of  Glucuronic  Acid  and  Glucosamin. 
The  mucins  occur  in  mucous  secretions,  as  for  example  the  secretions 
from  the  skin-gland  of  snails  or  slugs.  Mucoids  are  found  in  connec- 
tive tissues,  in  the  vitreous  humor  of  the  eye  and  in  the  white  of  egg 
(ovomucoid) .  The  chondroproteins  occur  especially  in  cartilaginous 
tissues,  and  in  the  interstitial  substance  of  connective  tissue.  Chondro- 
proteins are  also  found  in  the  accumulations  of  colloidal  material  which 
characterize  the  "amyloid  degeneration"  of  certain  organs  under 
pathological  conditions. 

Phosphoproteins. — The  phosphoproteins  are  proteins  which  yield 
phosphoric  acid  when  hydrolyzed.  The  most  typical  example  of  this 
group  is  Casein,  the  chief  protein  constituent  of  milk,  but  phospho- 
proteins also  occur  in  a  variety  of  vegetable  tissues,  and  in  the  yolk 
of  egg  (ovovitellin) .  They  are  predominantly  acid  in  character,  as 
might  be  expected,  not  only  from  their  content  of  phosphoric  acid,  but 
also  from  the  fact  that  they  yield  a  high  proportion  of  dicarboxylic 
amino-acids  on  hydrolysis. 

Chromoproteins. — The  chromoproteins  are  compound  proteins  in 
which  the  prosthetic  group  is  colored.  The  most  typical  examples  of 
the  group  are  Hemoglobin,  the  red  coloring-matter  and  oxygen-carrier 
of  blood,  in  which  the  prosthetic  group  is  a  complex  iron-containing 
organic  acid  Hematin,  and  Hemocyanin,  a  blue  pigment  containing 
copper  which  plays  a  role  corresponding  to  that  of  hemoglobin  in  the 
Arachnidce  and  Crustacea.  The  chromoproteins,  hemoglobin  and  hemo- 
cyanin,  are  exceptional  among  proteins  in  the  relative  ease  with  which 
they  are  obtainable  in  crystalline  condition.  The  protein  radical  in 
hemoglobin  is  a  predominantly  basic  protein,  known  as  Globin  and 
related  to  the  histones. 

Lecithoproteins. —  The  lecithoproteins  are  compound  proteins  in 
which  the  prosthetic  group  is  a  phospholipin.  This  is  rather  a  con- 
9 


130  THE  PROTEINS  AND  THE  AMINO-ACIDS 

jectural  group  of  substances,  for  although  proteins  associated  with 
phospholipins  have  been  prepared  from  yolk  of  egg,  and  from  vegetable 
tissues,  it  is  not  yet  certain  whether  the  phospholipin  is  an  integral 
part  of  the  protein  molecule,  or  merely  a  contamination  which  "is 
physically  adherent  to  it.  Evidence  of  an  electrochemical  character 
has  demonstrated,  however,  that  compounds  between  lecithin  and 
proteins  are  formed  when  the  two  substances  are  mixed  in  aqueous 
solution,  and  we  may  infer  that  similar  compounds  may  not  improbably 
exist  in  nature. 


HI.     THE  PRODUCTS  OF  PROTEIN  HYDROLYSIS. 

Infraproteins. — The  infraproteins  are  substances  produced  in  the 
initial  stages  of  protein  hydrolysis  which  still  retain  the  characteristic 
properties  of  the  proteins.  Examples  are  the  Acid-  and  Alkali-albumin- 
ates,  formed  from  albumins  by  gentle  heating  in  acid  or  alkaline  solu- 
tions, and  which  differ  from  albumins  in  being  insoluble  in  neutral 
distilled  water.  Other  examples  are  Paracasein,  formed  by  the  action 
of  rennet  or  weak  pepsin  upon  casein,  and  the  Paranucleins  which  are 
formed  by  the  partial  digestion  of  a  variety  of  phosphoproteins. 

Proteoses. — The  proteoses  are  hydrolytic  cleavage-products  of  the 
proteins  which  have  lost  the  characteristic  protein  property  of  being 
coagulable  by  heat,  but  they  retain  the  coagulability  by  ammonium 
sulphate.  They  are  usually  subdivided  into  Primary  Proteoses  which 
are  coagulable  by  half-saturation  of  their  solutions  with  ammonium 
sulphate,  and  Secondary  or  Deuteroproteoses  which  are  coagulated  by 
complete  saturation  of  their  solutions  with  ammonium  sulphate.  The 
majority  of  the  proteoses  are  coagulable  by  alcohol,  but  certain  of  them 
are  soluble  in  alcohol.  They  yield  a  reddish  violet  or  pink  biuret- 
reaction. 

A  considerable  number  of  the  proteoses  are  toxic  when  injected  into 
the  circulation,  while  the  native  proteins  with  a  few  marked  exceptions, 
such  as  the  Ricin  in  castor-oil  beans  (Ricinus  Communis)  are  non-toxic. 
On  the  other  hand  the  native  proteins  are  Antigenic  that  is,  they  give 
rise,  on  repeated  injection  into  the  circulation  of  animals,  to  sub- 
stances which  circulate  in  the  blood  serum  and  have  the  property  of 
precipitating  the  particular  protein  against  which  the  animal  has 
been  immunized.  The  proteoses  on  the  contrary  are  as  a  rule  non- 
ant  igenic. 

Peptones. — The  peptones  are  still  simpler  products  of  the  hydrolytic 
cleavage  of  proteins.  They  are  slightly  diffusible,  and  they  are  inco- 
agulable either  by  heat  or  by  ammonium  sulphate.  They  are,  however, 
precipitable  by  tannic  acid,  phosphotungstic  acid  or  lead  acetate. 
They  are  usually  coagulable  by  alcohol,  although  certain  peptones, 
especially  when  combined  with  acid,  are  not  coagulable  by  alcohol. 
They  yield  a  clear  pink  biuret-reaction,  and  are  non-antigenic  and,  as 
a  rule,  non-toxic. 


THE  END-PRODUCTS  OF  PROTEIN  HYDROLYSIS  131 


IV.     THE  COAGULATED  PROTEINS. 

The  coagulated  proteins  may  be  subdivided  into  two  classes,  namely, 
those  in  which  the  coagulation-process  has  gone  so  far  as  to  be  irrevers- 
ible, so  that  the  coagulum  cannot  be  brought  back  into  solution  again 
without  preliminary  decomposition  into  simpler  substances,  and  those 
in  which  the  coagulum  remains  soluble  after  removal  of  the  coagulating- 
agent  and  in  which  the  coagulation-process  has  therefore  remained 
reversible.  The  majority  of  the  heat-coagulated  proteins  belong  to  the 
first  class,  although  the  incipient  stages  of  heat-coagulation  are  some- 
times reversible.  On  the  other  hand  the  coagula  produced  by  alcohol 
or  by  ammonium  sulphate  belong  to  the  second  class,  although  in  some 
instances  after  more  or  less  prolonged  contact  with  alcohol  the  coagula 
produced  by  alcohol  become  irreversible. 

The  polypeptides  or  chains  of  amino-acids  out  of  which  proteins 
are  built  up,  form  anhydrides  with  exceptional  ease,  either  by  internal 
neutralization  of  carboxyl-  and  amino-groups,  or  by  the  condensation 
of  several  molecules,  and  this  tendency  increases  with  increasing 
length  of  the  amino-acid  chain.  We  can  hardly  suppose,  therefore, 
that  this  property  has  been  lost  in  the  much  more  bulky  amino-acid 
complexes  which  constitute  the  proteins.  On  the  other  hand  the 
agencies  which  bring  about  coagulation  are  all  of  such  a  character 
(heat,  alcohol  and  concentrated  salts)  as  to  suggest  that  the  with- 
drawal of  water  from  the  protein  is  the  chemical  basis  of  the  coagulation- 
process.  It  appears  very  probable,  therefore,  that  coagulation  is  due 
to  the  formation  of  protein  anhydrides,  and  that  the  irreversible 
coagula  are  those  in  which  the  anhydride-formation  has  proceeded 
furthest. 


THE    END-PRODUCTS    OF    PROTEIN    HYDROLYSIS: 
THE  AMINO-ACIDS. 

Decomposition  of  proteins  into  simple  crystal! izable  substances 
may  very  readily  be  brought  about  by  a  variety  of  agencies,  but  the 
only  methods  of  decomposition  which  yield  easily  interpretable  results 
are  those  which  bring  about  Hydrolysis,  or  decomposition  of  the  mole- 
cule by  successive  splittings  with  the  addition  of  the  elements  of 
water.  Hydrolysis  of  the  proteins  (autohydrolysis)  will  take  place 
spontaneously  in  neutral  protein  solutions  or  even  if  precipitated 
proteins  be  left  in  long-continued  contact  with  neutral  and  sterile 
water.  The  process  is,  however,  greatly  accelerated  by  the  application 
of  heat,  especially  by  temperatures  considerably  exceeding  the  tem- 
perature of  boiling  water,  or  by  catalyzers,  of  which  the  most  efficient 
are  acids,  alkalies  and  the  protein-digesting  (proteolytic)  enzymes. 
Whatever  the  means  of  hydrolysis  employed,  however,  the  end-result, 
provided  the  hydrolysis  has  been  complete,  is  the  same,  namely,  the 
production  of  a  mixture  of  amino-acids. 


132  THE  PROTEINS  AND  THE  AMINO-ACIDS 

Incomplete  hydrolysis,  however,  results  in  the  production  of  a 
number  of  intermediate  substances,  variously  designated,  in  the  order 
of  decreasing  complexity,  Proteoses  (Albumoses) ,  Peptones,  Polypep- 
tides  and  Dipeptides.  The  hydrolysis  of  the  proteins,  therefore,  occurs 
in  stages,  just  as,  in  the  hydrolysis  of  starch,  intermediary  stages  (the 
dextrins  and  maltose)  are  passed  through  before  the  attainment  of  the 
last  stage  of  hydrolysis  and  the  quantitative  conversion  of  the  starch 
into  glucose. 

It  is  not  certain,  however,  whether  the  various  intermediate  pro- 
ducts of  protein  hydrolysis  represent  successive  stages  of  hydrolysis 
or  whether  in  some  instances  comparatively  simple  products  may  not 
be  split  off  from  the  proteins  or  proteoses,  leaving  complex  residues, 
so  that  complex  and  simple  intermediate  substances  are  produced 
simultaneously.  Probably  both  types  of  cleavage  occur  at  different 
points  in  the  protein  molecule.  Those  linkages  which  are  most  acces- 
sible to  the  action  of  the  particular  catalyzer  employed  will  be  dis- 
rupted firstj  and  if  some  of  them  chance  to  lie  near  the  extremities  of 
the  molecule,  simple  products  and  a  complex  residue  will  result,  while 
disruption  of  more  internal  linkages  will  break  the  molecule  into  parts 
of  more  equal  weight  and  complexity. 

It  was  early  recognized  that  amino-acids  form  the  chief  part  of  the 
decomposition-products  which  result  from  the  hydrolysis  of  protein. 
The  separation  of  the  individual  amino-acids  from  one  another,  and 
their  quantitative  estimation,  was  a  much  more  difficult  matter.  The 
first  attempts  to  isolate  individual  amino-acids  from  the  mixture  which 
the  complete  hydrolysis  of  a  protein  yields,  depended  upon  the  frac- 
tional crystallization,  either  of  the  free  amino-acids  or  of  their  salts. 
Except  in  the  case  of  the  very  slightly  soluble  amino-acids,  such  as 
tyrosine,  these  methods  were  not  even  approximately  quantitative, 
and  even  the  isolation  and  identification  of  a  given  amino-acid  could 
only  be  effected  with  certainty  when  that  acid  was  present  in  relatively 
large  amounts.  The  attainment  of  our  present  relatively  extensive 
knowledge  of  the  nature  and  quantities  of  the  amino-acids  which 
result  from  protein  hydrolysis,  is  an  achievement  of  the  past  twenty 
years,  and  we  owe  it  in  the  first  place  to  the  investigations  of  Kossel 
and  of  Emil  Fischer  and  their  pupils. 

The  various  amino-acids  which  are  yielded  by  the  proteins  are  limited 
in  number,  and  probably  do  not  exceed  eighteen  or  nineteen.  These 
however,  fall  into  several  very  distinct  classes,  namely: 

Monoamino-monocarboxylic  acids,  general  formula: 

H2N.R.COOH 

Monoamino-dicarboxylic  acids,  general  formula: 

/coon 

H2N.R 


THE  END-PRODUCTS  OF  PROTEIN  HYDROLYSIS  133 

Diamino-monocarboxylic  acids,  general  formula: 

, 

\ 

R.COOH 

Diaminohydroxy-monocarboxylic  acids. 

Heterocyclic  compounds,  i.  e.,  amino-acids  containing  a  ring  of 
atoms,  one  or  more  nitrogen  atoms  being  included  in  the  ring. 

The  first  necessary  step  in  the  analysis  of  the  amino-acids  produced 
by  the  hydrolysis  of  protein,  consists  in  the  separation  of  the  diamino- 
acids  from  the  monoamino-acids.  This  may  be  accomplished  by  means 
of  phosphotungstic  acid,  which  precipitates  the  diamino-acids  while 
the  monoamino-acids  are  left  in  solution.  The  diamino-acids  Arginine 
and  Histidine  may  be  separated  in  the  form  of  their  silver  salts,  while 
the  remainder  of  the  precipitate  produced  by  phosphotungstic  acid 
consists  of  the  diamino-acid  Lysine. 

The  monoamino-acids  may  be  estimated  by  evaporating  the  whole 
mixture  to  dryness  in  vacuo  and  then  dissolving  the  mixture  of  acids 
in  alcohol,  and  passing  into  the  solution  dry  hydrochloric  acid  gas 
which  catalyzes  the  formation  of  alcohol  esters  of  the  amino-acids. 
These  esters  are  volatile  and  may  be  separated  into  fractions  each 
containing  only  a  small  number  of  acids,  by  means  of  fractional  dis- 
tillation in  vacuo.  The  esters  in  each  fraction  are  then  reconverted 
into  free  acids  and  alcohol  by  hydrolysis,  and  the  individual  acids 
separated  and  estimated  by  special  methods  adapted  to  the  peculiar 
properties  of  each  of  the  acids  present  in  the  particular  fraction  con- 
cerned. The  difficultly  soluble  acids,  Tyrosine,  Cysline  and  Diamino- 
trioxydodecanic  Acids  are  separated  from  the  digest  before  ester ification. 

This  method,  due  to  Emil  Fischer,  is  laborious  and  inaccurate,  but 
it  greatly  surpasses  the  methods  which  were  formerly  in  use,  and 
which  did  not  even  permit  a  partial  separation  of  the  various  mono- 
amino-acids in  a  protein  digest.  The  method  permits  the  accurate 
quantitive  determination  of  only  five  acids,  namely,  the  diamino-acids 
histidine,  arginine  and  lysine,  and  the  monoamino-acids  tyrosine  and 
cystine.  The  estimates  of  the  other  acids  are  only  approximate,  and 
must  be  regarded  as  minimum  values,  since  it  has  been  found  that  in  a 
known  mixture  of  amino-acids  it  is  only  possible  to  account  for  about 
two-thirds  of  the  nitrogen,  by  Fischer's  method.  For  many  purposes, 
in  which  a  knowledge  of  the  total  proportion  of  nitrogen  present  in  the 
form  of  monoamino-acids  suffices,  Fischer's  method  has  now  been 
largely  superseded  by  the  method  of  Van  Slyke  which  is  described 
below  (p.  144),  but  if  we  desire  to  ascertain  approximately  the  quantity 
of  the  individual  monoamino-acids  contained  in  a  protein  digest, 
Fischer's  method,  or  modifications  of  it,  still  affords  the  only  available 
procedure. 


134  THE  PROTEINS  AND  THE  AMINO-ACIDS 

if  By  these  various  methods  the  following  ammo-acids  have  been 
isolated  from  among  the  products  of  hydrolysis  of  various  proteins: 

A.  Monoamino-monocarboxylic  acids. 
1.  Glycine,  or  ammo-acetic  acid: 


2.  Alanine,  or  a-amino-propionic  acid: 

CH3.CH(NH2).COOH 

3.  Valine,  or  a-amino-iso-valerianic  acid: 

CHs 

CH.CH(NH2).COOH 
CH3 

4.  Leucine,  or  a-amino-isocaproic  acid: 

CH3 

CH.CH2.CH(NH2)  .COOH 
CH3 

5.  Isoleucine,  or  a-amino-/3-methyl-/3-ethylpropionic  acid  : 

CH3  \ 

CH.CH(NH2).COOH 
C2H6 

6.  Caprine,  or  Glycoleucine,  or  a-amino-normal-caproic  acid: 

CH3.CH2:CH2.CH2CH(NH2)  COOH 

7.  Phenylalanine,  or  0-phenyl-a-amino-propionic  acid  : 

C6H5CH2.CH(NH2)COOH 

8.  Tyrosine,  or  jS-parahydroxyphenyl-a-amino-propionic  acid: 

HO.C6H4.CH2.ciNH2).COOH 

9.  Serine,  or  jS-hydroxy-a-amino-propionic  acid: 

CH2(OH)  .CH(NH2)  .COOH 

10.  Cystine,  or  dicysteine  or  di-  (/3-thio-a-aniino-propionic  acid)  : 

HOOC.CH.(NH2).CH2S—  SCH2.CH(NH2).COOH 

V 


THE  END-PRODUCTS  OF  PROTEIN  HYDROLYSIS  135 

B.  Monoaminodicarboxylic  acids. 
11.  Aspartic  acid,  or  amino-succinic  acid: 

HOOC.CH2CH(NH2)  .COOH 

.    12.  Glutamic  acid,  or  a-aminoglutaric  acid: 

HOOC.CaHjCHaCHCNHiO.COOH 

C.  Diaminomonocarboxylic  acids. 

13.  Arginine,  or  a-amino-3-guanidine-valerianic  acid: 

xNH2 

HN    =  C 

NH,  .CH2CH2.CH(NH2)  .COOH 

14.  Lysine,  or  a-oj-diaminocaproic-acid : 

C  WvH2N.CH2.CH2.CH2.CH(NH2)  .COOH 

D.  Heterocyclic  amino-acids. 

.15.  Histidine,  or  /3-immazolyl-a-amino-propionic  acid: 


N  NH 

CH=    =C.CH2.CHCNH2).COOH 

*     16.  Proline,  or  pyrrolidine  carboxylic  acid: 

CH2  -       -  CH2 

CH2  CH.COOH 


•    17.  Oxyproline,  or  hydroxy-a-pyrrolidine-carboxylic  acid 
Either:' 

HOCH CH2 


CH2  CH.COOH 


or: 

CH2  -  CHOH 


CH2  CH.COOH 


18.  Tryptophane  or  /3-indole-o:-aminopropionic  acid 

C.CH2.CH.  (NH2)  .COOH 

/\ 

C6H4  CH 

/  \/ 

NH 


136  THE  PROTEINS  AND  THE  AMINO-ACIDS 

In  addition  to  these  acids  two  hydroxy-diamino-acids  have  been  iso- 
lated from  among  the  cleavage-products  of  one  protein,  namely,  casein. 
The  very  great  variety  of  proteins  and  protein  derivatives  which  exist 
in  nature  are  therefore  constructed  out  of  a  relatively  small  and  limited 
number  of  amino-acid  building-stones,  differing  proportions  and 
arrangements  of  these  components  being  responsible  for  the  wide 
variety  of  characteristics  displayed  by  the  native  and  derived  proteins. 

In  many  instances  a  definite  parallelism  can  be  traced  between  the 
chemical  and  physical  behavior  of  the  proteins  and  their  amino-acid 
content.  Thus,  the  Albumins,  which  are  soluble  in  distilled  water  and 
are  not  coagulated  by  half-saturation  of  their  solutions  with  am- 
monium sulphate,  contain  no  glycine,  while  the  Globulins,  which  (when 
uncombined  "with  acids  or  bases)  are  insoluble  in  distilled  water  and 
are  coagulated  by  half-saturation  of  their  solutions  with  ammonium 
sulphate,  do  contain  this  amino-acid.  The  alleged  transformation  of 
serum  albumin  into  globulin  by  warming  in  alkaline  solutions  observed 
by  Moll,  and  not  infrequently  quoted,  is  therefore,  an  impossibility, 
since  it  would  involve  the  synthesis  of  amino-acetic  acid  and  its  union 
with  the  albumin  molecule  which  could  not  be  brought  about  by  any 
such  simple  procedure.  The  product  actually  obtained  by  Moll  was 
an  infraprotein,  alkali-albuminate,  which  mimics  globulin  in  being 
insoluble  in  neutral  water,  but  differs  from  it  in  fundamental  consti- 
tution. 

The  alcohol-soluble  vegetable  proteins  (Prolamines)  contain  a  trace 
(probably  attributable  to  associated  impurities)  of  glycine,  and  some  of 
them  contain  no  glycine,  their  content  of  diamino-acids  is  very  small, 
while  their  content  of  glutamic  acid  and  of  proline  is  very  high.  The 
phosphoproteins,  Casein  and  Vitellin,  are  also  rather  high  in  glutamic- 
acid  content.  Gelatin  is  characterized  by  its  high  glycine  content  and 
Keratin  by  its  high  content  of  cystine.  The  Histones,  which  are  pre- 
dominantly basic  substances,  contain  about  thirty  per  cent,  of  diamino- 
acids,  while  the  Protamines,  which  are  still  more  predominantly  basic, 
contain  only  small  amounts  of  monoamino-acids,  Salmine  (from 
salmon  sperm)  containing  over  eighty  per  cent,  of  arginine,  while 
Sturine  (from  sturgeon  sperm)  contains  sixty-seven  per  cent,  of  its 
nitrogen  as  arginine,  ten  per  cent,  in  the  form  of  histidine,  and  from 
six  to  seven  per  cent,  in  the  form  of  lysine. 

/"   The  amino-acids  are  white,  crystalline,  readily  diffusible  substances 
/  and  the  crystal  form  is  characteristic  for  each  amino-acid.     The 
\  crystal  forms  of  glycine,   leucine  and   histidine  are  shown  in  the 
V  accompanying  figures  (3-5). 

The  amino-acids  are  usually  readily  soluble  in  water,  cystine  and 
tyrosine  affording  exceptions  to  this  rule.  They  are,  with  the  exceptions 
of  proline  and  oxyproline,  insoluble  in  alcohol  and  ether.  They  have 
high  melting-points  and  melt  with  decomposition,  splitting  off  carbon 
dioxide.  With  the  exception  of  glycine  the  amino-acids  are  optically 
active,  some  of  them  being  dextrorotatory  and  others  levorotatory. 


TH'E  END-PRODUCTS  OF  PROTEIN  HYDROLYSIS  137 


FIG.  3. — Glycine  crystals.     (After  Hawk.) 


FIG.  4. — Leucine  crystals.     (After  Funke.) 


k 


FTG.  5. — Histidine  monochloride  crystals. 


138  THE  PROTEINS  AND  THE  AMINO-ACIDS 

The  amino-acids,  since  they  contain  trivalent  nitrogen  and  a  car- 
boxyl-group,  are  simultaneously  bases  and  acids,  in  other  terminology 
are  Amphoteric  Acids.  They  form  crystalline  salts  with  metallic  bases 
and  with  mineral  acids. 

On  treatment  with  nitrous  acid  the  amino-acids  lose  their  amino- 
group,  which  is  replaced  by  a  hydroxyl-group  as  follows : 

CH2.NH2  CH2OH 

+     HNO2     =|  +     N2     +     H2O 

COOH  COOH 

It  will  be  observed  that  all  of  the  amino-acids  obtained  in  the 
hydrolysis  of  proteins  are  a-amino-acids,  that  is,  an  ami  no-group  is 
attached  to  the  carbon  atom  immediately  adjacent  to  the  carboxyl- 
group.  This  is  probably  a  fact  of  great  significance,  since,  as  we  shall 
see,  the  proteins  are  formed  by  the  union  of  long  chains  of  amino-acids, 
linked  together  by  means  of  their  amino-  and  carboxyl -groups.  These 
groups  being  closely  adjacent,  the  resultant  chains  are  shorter,  and  the 
weight  of  the  other  radicals  in  the  molecule  more  evenly  distributed 
than  would  be  the  case  if  the  carboxyl-  and  amino-groups  were  separated 
by  a  long  chain  of  carbon  linkages,  and  the  possibility  of  such  heavy 
compounds  as  the  proteins  possessing  sufficient  stability  to  permit 
their  formation  probably  resides  in  this  device  for  shortening  the  chain 
of  serial  linkages.  Corresponding  to  this  view  we  find  that  the  oo-amino- 
group  which  is  also  present  in  lysine,  is  not  united  in  proteins  to  any 
carboxyl-group  but  remains  free  and  reacts  with  nitrous  acid  just  as 
the  amino-acid  does. 

THE  SYNTHESIS  OF  PROTEINS. 

The  marked  predominance  of  amino-acids  among  the  products  of 
protein  hydrolysis  long  ago  led  biological  chemists  to  surmise  that  the 
amino-acid  structure,  or  some  derivative  of  that  structure,  must  be 
represented  in  a  high  degree  in  the  protein  molecule,  and  it  was  in 
following  up  this  clue  that  Schiitzenberger  in  1888  carried  out  one  of 
the  earliest  and  most  successful  attempts  to  synthesize  bodies  of  a 
protein  character.  Recognizing  that  the  decomposition  of  proteins 
into  amino-acids  is  essentially  a  phenomenon  of  hydrolysis,  he  regarded 
.dehydration  as  an  essential  feature  of  any  attempt  at  protein  syn- 
thesis, while  the  abundance  of  amino-acids  among  the  products  of 
protein  hydrolysis,  and  the  presence  therein,  as  he  thought,  of  bodies 
related  to  urea,  led  him  to  believe  that  protein  synthesis  must  consist 
in  the  linkage  of  amino-acids  with  molecules  of  urea  and  the  elimination 
of  water.  Accordingly  amino-acids  were  mixed  with  urea  and  phos- 
phorus p'entoxide  and  heated  to  125°  C.  The  product  was  a  pasty 
solid,  soluble  in  water  and  readily  coagulated  by  alcohol.  It  was 
furthermore  precipitated  from  aqueous  solutions  by  the  usual  protein 
precipitants  and  gave  the  biuret-  and  xanthoproteic  reactions. 


THE  SYNTHESIS  OF  PROTEINS  139 

This  experiment  of  Schiitzenberger's  left  us,  however,  very  much 
where  we  were,  so  far  as  real  knowledge  of  the  structure  of  the  protein 
molecules  is  concerned.  The  knowledge  of  the  fact  that  a  mixture  of 
amino-acids  and  urea  yields,  under  certain  treatment,  a  body  or  bodies 
more  or  less  closely  resembling  the  proteins,  furnished  us  with  little 
or  no  information  regarding  the  structure  of  the  protein  molecule 
which  we  did  not  already  possess  in  the  fact  that  the  disintegration- 
products  of  the  proteins  are  predominantly  amino-acids.  Prior  to 
Schiitzenberger,  Grimaux,  in  1881,  had  shown  that  condensatioa- 
products  of  aminobenzoic  acid  and  aspartic  acid  resemble  the  proteins 
in  many  of  their  properties;  but  these  experiments  also  threw  no  light 
upon  the  structure  of  the  protein  molecule  beyond  emphasizing  the 
already  sufficiently  evident  probability  that  the  amino-acid  grouping 
plays  an  important  part  in  the  building  up  of  the  protein  molecule. 

The  clue  which  led,  through  a  series  of  remarkable  researches,  to 
our  present  comparatively  extensive  knowledge  of  the  groupings 
within  the  protein  molecule,  was  obtained  in  1883  by  Curtius  when 
he  discovered  that  ethyl  glycocollate  (the  ethyl  ester  of  glycine)  in 
watery  solution  tends  to  form  Glycine  Anhydride: 

(In  the  absence  of  water) 

H2N.CH2.COOH     +     C2H6OH     =     H2N.CH2COOC2H6     +     H2O 

(Glycine)  +  (Ethyl  alcohol)  =  (Ethyl  glycocollate). 

(In  the  presence  of  water). 

H2N.CH2COOC2H6      +     Hj-N.CH^COOCzHs     = 

(Ethyl  glycocollate)         +        (Ethyl  glycocollate) . 

CH2 N  HL 

O  =  C  C    =  O     +     2C2H6OH 

^NH CH/ 

(Glycine  anhydride)  +  (Ethyl  alcohol) . 

Obviously,  if  the  closed  ring  representing  the  glycine  anhydride 
molecule  could  be  opened  up  without  destroying  the  stability  of  the 
molecule,  a  new  amino-acid  would  be  formed,  one  degree  more  complex 
than  the  original  amino-acid  (glycine) .  This  possibility  was  realized 
by  Emil  Fischer,  who  found  that  if  the  glycine  anhydride  which  is 
thus  prepared  be  boiled  for  a  short  time  with  concentrated  hydro- 
chloric acid,  the  following  change  occurs: 

/CH2.NHx  /CH?.NH2.HC1 

O  =  C  C  -  O  +  HC1  +  H2O  =  O  = 

\  / 

(Glycine  anhydride.)  (Glycyl-glycine  chloride.) 


140  THE  PROTEINS  AND  THE  AMINO-ACIDS 

On  now  treating  the  glycyl-glycine  chloride  with  silver  oxide,  silver 
chloride  is  precipitated  and  free  Glycyl-glycine  is  obtained.  If,  how- 
ever, the  glycine  anhydride  be  originally  treated  with  Alcoholic  instead 
of  with  an  aqueous  solution  of  hydrochloric  acid,  the  ethyl  ester  of 
glycyl-glycine  is  obtained: 

xCH2.NHv  /CH2NH2 


C  C  =  O  +  C*H6OH    =    O=C 

\  /  \ 


(Glycine  anhydride.)  (Glycyl-glycine  ester.) 

It  would  appear,  therefore,  as  if  we  had  only  to  repeat  this  cycle 
of  operations  indefinitely  in  order  to  secure  the  most  complex  poly- 
amino-acids;  but  this  is  not  so  easy  as  it  might  appear  at  first  sight; 
the  instability  of  polyamino-acids  consequent  upon  the  high  reactivity 
of  the  —  NH2  group,  and  the  consequent  difficulty  of  obtaining  simple 
anhydrides  renders  this  procedure  impossible.  Moreover  the  anhy- 
dride-ring is  in  many  cases  (e.  g.,  leucine  anhydride)  very  difficult  to 
break  up  when  it  has  once  been  formed. 

In  the  search  for  methods  of  overcoming  these  difficulties  Fischer 
found  that  the  instability  of  the  amino-acids  could  be  eliminated  by  the 
introduction  of  radicals  into  the  —  NH2  group,  and  he  and  Fourneau 
synthesized  phenylcyanate-glycyl-glycine  (C6H5.NH.CO-NHCH2CO 
-NHCH2COOH)  and  carboxethyl-glycyl-glycine  ester  (C2H5O.OC. 
NH.CH^CO-NH.CH^COOC,!^  which  are  both  chemically  stable 
bodies.  In  subsequent  investigations  Fischer  found  that,  by  gentle 
heating,  combination  between  the  esters  of  the  carboxethyl-amino- 
acids  and  other  amino-acids  could  be  directly  brought  about,  and  in 
this  way  carboxethyl-diglycyl-leucine  ester  was  formed: 

C2H6OOC.NH.CH2CO.NH.CH2.CO.NH.CH.(C4H9)CO.OC2H5. 

The  difficulty  was  here  encountered,  however,  that  the  carboxethyl- 
group  having  been  once  introduced,  cannot  be  eliminated  again. 

The  method  which  was  devised  to  overcome  this  difficulty  was 
extremely  ingenious.  The  introduction  of  a  radical  into  the  —  NH2 
group  appeared  to  be  a  necessity,  forced  upon  us  by  the  impossibility 
of  otherwise  securing  simple  anhydrides  of  the  acids.  It  occurred  to 
Fischer,  however,  that  the  radical  thus  introduced  into  the  —  NH2 
group  might  itself  be  made  a  carrier  of  amino-acid  groups  into  the 
molecule.  This  anticipation  proved  to  be  correct.  The  radical  which 
was  first  utilized  was  the  chloracetyl  group  (C1CH2.CO—  ).  When 
chloracetyl  chloride  is  allowed  to  act  upon  glycyl-glycine  ester  (ob- 
tained by  the  methods  described  above),  chloracetyl-glycyl-glycine- 
ester  is  obtained: 

C1CH2COC1   +  H2N.CH2CO.NH.CH2COOC2H6      = 
Chloracetyl  chloride)      +     (Glycyl-glycine  ester)         = 

C1CH2.CO.NH.CH2.CO.NH.CH2.COOC2H5  +  HC1 

(Chloracetyl  glycyl-glycine  ester)  . 


THE  SYNTHESIS  OF  PROTEINS  .   141 

By  saponification  of  this  ester,  free  chloracetyl-glycyl-glycine  is 
obtained.  On  now  treating  this  with  a  concentrated  aqueous  solution 
of  ammonia,  the  chlorine  atom  in  the  chloracetyl  group  becomes,  by 
a  usual  reaction,  replaced  by  an  amino-group,  and  thus  Diglycyl-glycine 
is  obtained: 

C1CH2.CO.NH.CH2.CONH,CH2COOC2H5     +     2NH3      = 
(Chloracetyl  glycyl-glycine  ester) 

H2N.CH2.CONH.CH2CONH.CH2COOC2H5     +     NEUC1 

(Diglycyl-glycine  ester). 

In  other  words,  the  chloracetyl-group,  introduced   to  protect  the 

—  NH2-group  of  the  amino-acid  is,  after  it  has  performed  its  protective 
function,  itself   transformed  into  an  amino-acid-group,   through  the 
replacement  of  the  halogen  atom  by  —  NH2.  Obviously,  other  halogen- 
containing  acid  groups  may  be  used  in  place  of  chloracetyl,  and  in  this 
way  a  great  variety  of  amino-acid-groups  can  be  introduced  into  the 

—  NH2-group.    Among  others  the  following  are  employed: 
Chloracetyl  chloride  for  the  introduction  of  glycyl. 
a-Bromopropionyl  chloride  for  the  introduction  of  alanyl. 
a-Bromisocapronyl  chloride  for  the  introduction  of  leucyl. 
a-Phenylbromopropionyl  chloride  for  the  introduction  of  phenyl- 

alanyl. 

a-S-Dibromovaleryl  chloride  for  the  introduction  of  prolyl. 

By  this  method  the  chain  of  amino-acids  is  lengthened  at  the  amino- 
group  end.  Theoretically  it  appeared  possible  to  also  lengthen  the 
chain  at  the  carboxyl-end  of  the  molecule,  by  acting  upon  the  esters 
of  the  amino-acids  with  the  acid  chlorides  of  other  amino-acids.  Until 
1904,  however,  the  acid  chlorides  of  amino-acids  were  unknown,  and 
all  attempts  to  prepare  them  had  failed,  owing  to  the  same  reason 
which  limits  the  use  of  the  first  method  of  synthesizing  poly-amino- 
acids  described  above,  namely  the  reactivity  of  the  —  NH2-group.  It 
will  be  recollected  that  Fischer  found  that  the  —  NH2-group  could  be 
protected  by  the  introduction  of  radicals,  and,  utilizing  this  fact,  in 
1904  he  succeeded  in  devising  a  method  of  preparing  the  acid  chlorides 
of  the  amino-acids.  The  acid  chlorides  thus  prepared,  react  with  the 
esters  of  other  amino-  or  polyamino-acids  to  form  polyamino-acid 
chains  of  greater  length.  Thus: 

C4H9CHBr.CO.NH.CH2.COCl     +     2NH2.CH2.COOC2H5      = 

(Bromisocapronyl-glycyl  chloride)  Glycine  ester) 


+     C4H9CHBrCONH.CH2CO.NH.CH2COOC2H5 
(Glycine  ester  hydrochloride)  (Bromisocapronyl-glycyl-glycine  ester) 

Subsequent  saponification  of  the  bromisocapronyl-glycyl-glycine 
ester  and  treatment  with  ammonia  yields  the  polyamino-acid  or 
(tripeptide)  ,  Leucyl-glycyl-glycine  : 

aH9CH(NH2)CO.NH.CH2CONH.CH2.COOH 


142  THE  PROTEINS  AND  THE  AMINO-ACIDS 

If  the  bromisocapronyl-glycyl  chloride  be  made  to  act  upon  glycyl- 
glycine  ester,  and  the  product  treated  with  ammonia,  the  tetrapeptide 
Leucyl-diglycyl-glycine  results: 

C4H9.CH(NH2)CO.NHCH2.CO.NHCH2CO.NHCH2COOH 

By  these  methods,  and  modifications  of  these  methods,  Fischer  and 
others  have  succeeded  in  building  up  long  chains  of  amino-acid-groups, 
these  chains  being  collectively  termed  by  Fischer,  Peptides.  Chains 
consisting  of  two  links,  i.  e.,  combinations  of  two  amino-acids  are 
designated  Dipeptides;  such,  for  example,  are  glycyl-glycine,  alanyl- 
alanine  and  leucyl-leucine,  chains  consisting  of  three  links  are  termed 
Tripeptides,  such  being,  for  example,  diglycyl-glycine  and  leucyl-glycyl- 
glycine.  Chains  consisting  of  four  links  are  termed  Tetrapeptides, 
and  so  on,  the  higher  members  of  the  series  being  collectively  termed 
Polypeptides. 

The  surpassing  interest  of  these  investigations  lies  in  the  fact  that 
many  of  the  polypeptides  are  considered  to  be,  in  all  probability, 
identical  with  certain  of  the  natural  peptones  derived  from  proteins  by 
partial  hydrolysis,  while  others  probably  merit  inclusion  among  the 
proteins  themselves.  Thus  the  Octadecapeptide,  1-leucyl-triglycyl- 
1-leucyl-triglycyl-l-leucyl-octaglycyl-glycine,  and  the  Tetradecapeptide, 
1-leucyl-triglycyl-l-leucyl-octaglycyl-glycine  so  closely  resemble,  in 
general  properties,  the  ordinary  proteins,  that  they  would  undoubtedly 
have  been  classed  among  the  proteins  had  they  been  first  met  with  in 
nature.  Thus,  they  give  the  biuret-reaction,  and  form  opalescent 
watery  solutions,  and  the  tetradecapeptid  is  coagulated  by  ammonium 
sulphate  and  precipitated  by  tannic  acid  and  by  phosphotungstic  acid. 
As  they  do  not  contain  tyrosine,  tryptophane,  phenylalanine  or 
cystine,  they  fail  to  give  such  protein  color  reactions  as  depend  upon 
the  presence  of  these  groups. 

The  molecular  weight  of  the  octadecapeptide  is  1213,  and  the  sub- 
stitution of  phenylalanine,  tyrosine  and  cystine  in  the  place  of  glycine 
groups  would  increase  this  weight  two  or  three  times,  giving  a  value 
which  is  of  the  same  order  of  magnitude  as  the  more  modern  esti- 
mations of  the  (minimal)  molecular  or  combining-weights  of  many  of 
the  natural  proteins. 

A  whole  series  of  the  polypeptides  give  the  typical  peptone  biuret 
reaction  (pink),  and  such  as  contain  tyrosine  also  give  Millon's  reac- 
tion. The  biuret-reaction  is,  with  the  glycine  compounds,  first  encoun- 
tered in  the  tetrapeptide,  but  it  is  given  by  tripeptides  built  up  to 
include  other  amino-acids.  The  biuret-reaction  is,  generally  speaking, 
more  intense  the  greater  the  length  of  the  polypeptide  chain. 

The  majority  of  the  polypeptides  are  readily  soluble  in  water,  and 
such  as  are  soluble  in  water  with  difficulty,  are  readily  soluble  in  dilute 
mineral  acids  and  alkalies,  with  which  they  combine;  they  are  less 
soluble  in  solutions  of  acetic  acid.  As  a  rule  they  are  insoluble  in 
absolute  alcohol,  but  in  alcohol  containing  a  little  watery  ammonia 


PEPTIDES  AMONG  PROTEIN  HYDROLYSIS  143 

they  may  be  soluble,  in  which  case  they  are  precipitated  on  boiling  off 
the  ammonia. 

Under  conditions  involving  dehydration,  as  for  example  heating,  or 
treatment  of  the  esters  with  alcoholic  ammonia,  the  dipeptides  are 
converted  into  anhydrides  which  are  ring-compounds,  designated 
Dikelopiperazines,  and  having  the  general  formula: 

/CH.R.CCK 

NH</  NNH 

NDO.R.CH/ 

Under  similar  conditions  the  polypeptides  are  modified  in  an 
analogous  manner,  yielding  anhydrides  with  a  ring-structure. 

When  hydrolyzed,  the  polypeptides  break  down  into  their  constit- 
uent amino-acids,  the  imino-groups  in  the  polypeptide  molecule  being 
converted,  by  taking  up  the  elements  of  water,  into  amino-groups,  the 
reaction  being: 

-COHN-    +  H2O  =    -COOH  -f  H2N- 

A  very  large  number  of  the  polypeptides  are  hydrolyzed  by  the  pro- 
tein-splitting enzymes,  Pepsin,  Trypsin,  etc.,  and,  in  some  cases,  at  all 
events,  it  is  certain  that  the  hydrolysis  takes  place  in  stages,  as  it  does 
with  the  proteins  and  peptones.  We  will  revert  in  more  detail  to  this 
question  in  a  later  chapter  (Chapter  X) .  . 

THE  OCCURRENCE  OF  PEPTIDES  AMONG  THE  PRODUCTS 
OF  PROTEIN  HYDROLYSIS. 

The  fact  that  hydrolyzing  agents,  when  they  act  upon  proteins, 
lead  to  the  production  of  the  same  substances,  amino-acids,  that  the 
polypeptides  yield  when  hydrolyzed,  and  the  fact  that  the  synthetic 
polypeptides  resemble  the  native  and  derived  proteins  so  closely  in 
their  physical  and  chemical  characteristics,  afford  a  very  strong  basis 
for  the  supposition  that  the  proteins  are,  in  fact,  chains  of  ammo-acid 
radicals,  linked  together  as  they  are  in  the  polypeptides  by  the  mutual 
neutralization  of  — NH2-  and  — COOH-groups.  Confirmation  of  this 
view  is  afforded  by  the  fact  that  di,-  tri-  and  tetrapeptides  have  been 
frequently  found  and  identified  among  the  products  of  the  incomplete 
hydrolysis  of  proteins.  Thus  glycyl-d-alanine,  glycyl-1-tyrosine,  and 
a  tetrapeptide  built  up  by  the  union  of  two  molecules  of  glycine,  one 
of  d-alanine  and  one  of  1-tyrosine  have  been  isolated  from  among  the 
cleavage-products  of  Fibroin,  glycyl-1-leucine,  1-leucyl-d-alanine,  glycyl- 
d-valine  and  a  dipeptide  yielding  d-alanine  and*  1-proline  on  hydrolysis, 
have  been  obtained  from  Elastin,  glycyl-1-proline  has  been  obtained  from 
Gelatin,  and  a  lysyl-glycyl  peptide  from  Egg-albumin.  From  Gliadin 
a  dipeptide  has  been  split  off  which  yields  phenylalanine  and  proline 
when  completely  hydrolyzed. 


144  THE  PROTEINS  AND  THE  AMINO-ACIDS 

From  the  construction  of  these  fragments  of  the  protein  molecule 
we  may  infer  the  architecture  of  the  whole,  and  without  going  so  far 
as  to  assume  that  no  other  types  of  linkage  exist  within  the  protein 
molecule,  an  assumption  which  would  very  possibly  be  incorrect,  we 
may  safely  conclude  that  the  — COHN-linkage  plays  a  very  predomi- 
nant part  in  building  together  the  constituent  parts  of  the  protein. 

The  tetrapeptide  referred  to  above  which  has  been  isolated  from 
the  products  of  the  incomplete  hydrolysis  of  silk  fibroin  is  of  especial 
interest,  because,  had  it  not  been  identified  as  a  tetrapeptide,  it  would 
certainly  have  been  included  among  the  proteoses.  It  is  precipitated 
by  phosphotungstic  acid,  readily  dissolves  in  water,  is  insoluble  in 
alcohol,  and  coagulated  by  saturation  of  its  aqueous  solutions  with 
ammonium  sulphate,  or  with  sodium  chloride.  Its  molecular  weight, 
determined  by  the  cryoscopic  (freezing-point)  method,  was  350.  The 
synthetic  pentapeptide,  1-leucyl-triglycyl-l-tryosine,  'possesses  very 
similar  properties,  so  that  the  proteoses  are  not  necessarily  exceedingly 
complex  substances,  nor  is  excessive  complexity  necessary  in  order 
that  substances  of  this  type  may  be  coagulable  by  saturation  of  their 
aqueous  solutions  with  ammonium  sulphate. 

THE  ANALYSIS  AND  CHARACTERIZATION  OF  PROTEINS  BY 

THE  DETERMINATION  OF  THE  AMINO-ACID  RADICALS 

WHICH  THEY  CONTAIN. 

The  hydrolysis  of  the  proteins  is  accompanied  by  a  very  marked 
increase  in  the  total  number  of  free  amino-groups  present  in  the  solu- 
tion. This  is  due  to  the  fact  that  successive  repetitions  of  the  reaction : 

— COHN h  H2O   =  — COOH   +  H2N— 

result  in  the  transformation  of  imino-groups  into  amino-groups,  until 
the  final  number  of  amino-groups  formed  corresponds  \vith  the  total 
number  of  amino-acid  radicals  out  of  which  the  protein  is  built  up. 

Reference  has  been  made  above  to  the  fact  that  free  amino-groups 
in  the  aliphatic  series  have  the  well-known  property  of  reacting  with 
nitrous  acid,  with  the  liberation  of  nitrogen,  in  accordance  with  the 
equation: 

— RNH2   +  HNO2   =  ROH    +  H2O  +  N2 

Very  ingenious  advantage  has  been  taken  of  this  fact  by  D.  D.  Van 
Slyke,  in  the  method  which  he  has  devised,  and  which  is  now  very 
widely  utilized  for  the  determination  of  the  distribution  and  partition 
of  nitrogen  within  the  protein  molecule. 

This  method  consists  essentially  in  the  following  process:  The 
protein  having  been  in,the  first  place  subjected  to  complete  hydrolysis, 
the  small  proportion  of  ammonia  which  always  occurs  in  the  mixture  of 
products  (derived  from  "amide"  nitrogen  in  the  protein  molecule)  is 
first  removed  by  vacuum-distillation  and  separately  determined.  The 


ANALYSIS  AND  CHARACTERIZATION  OF  PROTEINS          145 

residual  mixture  of  products  is  then  treated  with  phosphotungstic  acid, 
which  results  in  the  precipitation  of  the  diamino-acids,  namely  Cystine, 
Arginine,  Lysine  and  Histidine,  a  determination  of  sulphur  yields  a 
measure  of  the  cystine-content.  Arginine  has  the  property  of  yielding 
one-half  of  its  nitrogen  in  the  form  of  ammonia  on  boiling  with  alkali. 
The  quantity  of  ammonia  developed  on  boiling  the  precipitate  with 
alkali  therefore  affords  a  measure  of  the  content  of  arginine. 

The  total  nitrogen  in  the  phosphotungstic-acid  precipitate  is  now 
determined,  and  from  it  is  subtracted  the  calculated  proportion  of 
nitrogen  which  is  contributed  by  the  cystine-  and  arginine-content. 
The  residual  nitrogen  is  derived  from  lysine  (=x)  and  histidine  (=y). 
On  treatment  with  nitrous  acid  lysine  yields  a  volume  of  free  nitrogen 
corresponding  to  the  whole  of  its  nitrogen-content  (=x),  while  histidine 
yields  a  volume  of  free  nitrogen  which  corresponds  to  one-third  of 
its  nitrogen-content  (=Jy).  The  amino-nitrogen  content  of  the 
precipitate  is  therefore  determined,  by  the  nitrogen-yield  on  treatment 
with  nitrous  acid,  and  after  subtracting  the  amino-nitrogen  yield  due  to 
arginine  (=  one-fourth  of  the  total  nitrogen  in  arginine)  and  of  cystine 
(=  the  whole  of  its  nitrogen  content)  the  residual  amino-nitrogen 
evidently  represents  the  whole  of  the  lysine  nitrogen  plus  one-third  of 
the  histidine  nitrogen,  or  x-fjy.  But  the  determination  of  the  total 
nitrogen  in  the  phosphotungstic-acid  precipitate,  and  the  subtraction 
therefrom  of  the  cystine  and  arginine  nitrogen,  has  already  given  us  a 
measure  of  the  total  nitrogen  contained  in  the  lysine  and  histidine, 
that  is,  of  x-f-y.  Subtracting,  therefore,  the  amino-nitrogen  yield  due 
to  these  two  amino-acids  from  the  proportion  of  the  total  nitrogen 
which  they  contribute,  the  difference  evidently  corresponds  to  two- 
thirds  of  the  histidine  nitrogen,  from  which  the  contents  of  histidine 
and  lysine  may  be  readily  computed. 

In  the  filtrate  from  the  phosphotungstic-acid  precipitate  the  total 
nitrogen  and  the  amino-nitrogen  are  separately  determined.  The 
difference  yields  a  measure  of  the  nitrogen  contained  in  pyrrolidine 
(proline  and  oxyproline)  or  indole  (tryptophane)  rings. 

For  the  determination  of  the  nitrogen  evolved  from  amino -groups 
on  treatment  with  nitrous  acid  Van  Slyke  has  devised  a  very  convenient 
apparatus  which  permits  the  rapid  and  accurate  determination  of  amino- 
groups  in  very  small  quantities  of  material.  For  descriptions  of  this 
apparatus  the  reader  is  referred  to  Van  Slyke's  original  articles,  or  to 
current  laboratory-handbooks.1 

During  the  hydrolysis  of  proteins  by  hydrochloric  acid  a  small 
amount  of  a  very  deeply  colored  precipitate  separates  out.  The 
nitrogen-content  of  this  precipitate  is  the  so-called  "humin"  nitro- 
gen. This  appears  to  be  derived  from  a  portion  of  the  tryptophane 
and,  in  the  presence  of  a  sufficiency  of  carbohydrate,  which  acts  as  a 
catalyzer  for  the  formation  of  humin,  the  yield,  of  humin-nitrogen  is 

1  For  example  R.  H.  A.  Plimmer:     Practical  Organic  and  Biochemistry,  London, 
1915,  p.  146. 
10 


146 


THE  PROTEINS  AND  THE  AMINO-ACIDS 


stated  to  be  a  quantitative  measure  of  the  tryptophane-content  of  the 
protein. 

The  following  are  representative  results  obtained  by  Van  Slyke's 
method. 

PERCENTAGE  OF  THE   TOTAL  NITROGEN  CONTAINED  IN  VARIOUS  AMINO- 
ACID   GROUPS   OF  THE   UNDERMENTIONED  PROTEINS. 


Gliadin. 

Edestin. 

Keratin 

(dog's  hair). 

Gelatin. 

Fibrin. 

Haemo- 
globin. 

Ammonia  N. 

25.52 

9.99 

10.05 

2.25 

8.32 

5.2 

Humin  N.     . 

0.86 

1.98 

7.42 

0.07 

3.17 

3.6 

Cystine  N.    . 

1.25 

1.49 

6.60 

0.00 

0.99 

0.0 

Arginine  N.  . 

5.71 

27.05 

15.33 

14.70 

13.86 

7.7 

Histidine  N.  . 

5.20 

5.75 

3.48 

4.48 

4.83 

12.7 

Lysine  N. 

0.75 

3.86 

5.37 

6.32 

11.51 

10.9 

Mono-amino  N. 

51.98 

47.55 

47.50 

56.30 

54.30 

59.0 

Non-amino  N. 

8.50 

1.70 

3.10 

14.90 

2.70 

2.9 

(  =  proline    + 

oxyproline  + 

^  tryptophane 

N) 

Total     .    '. 

99.70 

99.37 

98.85 

99.02 

99.58 

100.04 

The  following  are  among  the  estimations  of.  the  amino-acids  yielded 
by  various  proteins,  estimated  by  the  more  exhaustive  but  less  con- 
venient method  of  Emil  Fisher.1  It  must  be  recollected,  however, 
that  the  estimates  for  the  majority  of  the  monamino-acids  are  minimal 
estimates  only,  for  the  reason  that  in  the  process  of  analysis  a  certain 
proportion  of  the  nitrogen  escapes  estimation.  It  is  probable,  however, 
that  in  experienced  hands  the  losses  are  of  similar  magnitude  and  kind 
so  that  the  results  obtained  by  different  investigators  may  be  con- 
sidered comparable.  From  these  figures  it  may  clearly  be  seen  how 
variable  is  the  composition  of  different  proteins  in  respect  to  their 

1  Cited  from  analyses  reported  by: 

Osborne,  T.  B.,  and  Mendell,  L.  B.:     Jour.  Biol.  Chem.,  1914,  17,  p.  336. 

Osborne,  T.  B.,  and  Jones:     Am.  Jour.  Physiol.,  1910,  26,  p.  305. 

Kossel:     Zeit.  fur  Physiol.  Chem.,  1905,  44,  p.  347. 

Osborne  and  Guest:     Jour.  Biol.  Chem.,  1911,  9,  p.  425. 

Osborne  and  Clapp:     Am.  Jour.  Physiol.,  1906-7,  17,  p.  231. 

Osborne,  Van  Slyke,  Leavenworth  and  Vinograd:     Jour.  Biol.  Chem.,  1915,  22, 

p.  259. 

Abderhalden,  E.:     Zeit.  Physiol.  Chem.,  1902-3,  37,  p.  499. 
Abderhalden,  E.:     Zeit.  Physiol.  Chem.,  1903-4,  40,  p.  249. 
Kossel,  A.,  and  Patten,  A.  J.:     Zeit.  Physiol.  Chem.,  1903,  38,  p.  39. 
Fischer,  E.:     Zeit.  Physiol.  Chem.,  1903,  39,  p.  155. 
Morner:     Ibid.,  1901-2,  34,  p.  207. 

Osborne  and  Guest:     Jour.  Biol.  Chem.,  1911,  9,  p.  333. 
Abderhalden  and  Pribram:     Zeit.  Physiol.  Chem.,  1907,  51,  p.  409. 
Fischer  and  Bolhner:     Zeit.  Physiol.  Chem.,  1910,  65,  p.  118. 
Fischer,  Levene  and  Aders:     Zeit.  Physiol.  Chem.,  1902,  35,  p.  70. 
Skraup  and  Biehler:     Monatsh.  f.  Chem.,  1909,  30,  p.  467. 
Levene  and  Beatty:     Zeit.  Physiol.  Chem.,  1906,  49,  p.  252. 
Taylor,  A.  E.:     Jour.  Biol.  Chem.,  1908-9,  5,  p.  389. 
Osborne  and  Jones:    Am.  Jour.  Physiol.,  1909,  24,  p.  437. 


ANALYSIS  AND  CHARACTERIZATION  OF  PROTEINS 


147 


content  of  the  various  ammo-acid  radicals  and  how  great  a  rearrange- 
ment and  reassortment  of  radicals  is  necessary  before  one  protein  can 
be  converted  into  another  and,  consequently,  before  the  various 
proteins  of  the  diet  can  be  converted  into  the  characteristic  proteins  of 
the  various  tissues  of  the  body. 


PROPORTION  OF  VARIOUS  AMINO-ACIDS  YIELDED  BY  CERTAIN   PROTEINS 

ON  HYDROLYSIS. 


'a? 

? 

2" 

a 

§«» 

^  G 

«"c' 

•g-g 

's  „•§' 

-g 

"H"^' 

rS*"' 

Amino  acid. 

11 

£3  go 

v^« 

J^« 

ao 

11  8 

|    8 

G  a 

?Sa 

1|| 

'3    OJ 

l'J,ft 

cj  Q) 
0}   P* 

S     1, 

'1 

3 

'ST. 

6 

3 

0 

PH 

0 

Glycine     

0.0 

0.0 

3.8 

0.0 

0.0 

16.5 

0.0 

2.1 

Alanine     .       .       .    '  . 

9.8 

2.0 

3.6 

1.5 

2.5 

0.8 

0.0 

3.7 

Valiiie       .... 

1.9 

3.4 

6.2 

7.2 

0.9 

1.0 

5.3 

0.8 

Leucine     .... 

19.6 

6.6 

20.9 

9.4 

19.4 

2.1 

0.0 

11.7 

Proline      .... 

9.0 

13.2 

4.1 

6.7 

4.0 

7.7 

10.8 

5.8 

Oxyproline     ... 

1 

? 

2.0 

0.3 

6.4 

0.0 

Phenylalanine 

6.6 

2.4 

3.1 

3.2 

2.4 

0.4 

0.0 

3.2 

Aspartic  acid 

1.7 

0.6 

4.5 

1.4 

1.0 

1.2 

0.0 

4.5 

Glutamic  acid 

26.2 

43.7 

18.7 

15.6 

10.1 

1.8 

0.0 

15.5 

Serine        .... 

1.0 

0.2 

0.3 

0.5 

? 

0.4 

8.7 

? 

Tyrosine  .... 

3.6 

1.2 

2.1 

4.5 

0.9 

0.0 

0.0 

2.2 

Cystine     .      .      .      . 

? 

0.5 

0.3 

0.1 

? 

0.0 

0.0 

Histidine 

0.8 

2.2 

2.4 

2.5 

2.1 

0.4 

0.0 

1.8 

Arginine   .... 

1.6 

3.0 

14.4 

3.8 

3.2 

7.6 

91.7 

7.5 

Lysine       .... 

0.0 

1.2 

1.7 

6.0 

9.2 

2.8 

0.0 

7.6 

Tryptophane  . 

0.0 

1.0 

1.5 

0.0 

0.0 

Ammonia. 

3.6 

5.2 

2.3 

1.6 

1.3 

1.1 

REFERENCES. 

GENERAL  CHARACTERISTICS  OF  THE  PROTEINS: 

Robertson:     The  Physical  Chemistry  of  the  Proteins.     New  York,  1918. 
HYDROLYSIS  AND  SYNTHESIS  OF  PROTEINS  AND  POLYPEPTIDES: 

Fischer:     Untersuchungen  iiber   Aminosauren,   Polypeptide   und  Proteine    (1899- 

1906).     Berlin,  1906. 

Plimmer:     The  Chemical  Constitution  of  the  Proteins.     3  parts.     London,  1917. 
Van  Slyke:     Jour.  Biol.  Chem.,  1909-10,  7,  p.  xxxiv;  1911,  9,  p.  185;  1911-12,  10, 
p.  15;  1915,  23,  p.  411;  1912,  12,  p.  295;  1913-14,  16,  pp.  121  and  125;  1915,  22, 
p.  281;  1915,23,  p.  407. 


CHAPTER  VIII. 
COMPOUNDS  OF  THE  PROTEINS. 

TYPES  OF  UNION  IN  THE  PROTEIN  MOLECULE. 

Following  the  recognition  of  the  fact  that  the  proteins  are  complexes 
built  up  by  the  union  of  amino-acids,  the  question  of  the  mode  of  union 
between  them  became  one  of  paramount  importance.  Hofmeister 
has  pointed  out  that  it  is  possible  to  conceive  of  several  ways  in 
which  amino-acids  might  be  linked  together,  such  as : 
A.  Direct  union  of  the  carbon  atoms,  as: 


under  which  condition  the  molecule  would  be  an  immense  chain  of 
carbon  atoms  and  if  the  addition  of  the  elements  of  water  (hydrolysis) 
were  to  accomplish  the  splitting  up  of  the  molecule,  a  large  proportion 
of  hydroxy-acids  would  result: 

+     H.OH     =     — CH     +     HOC— 

I  I 

whereas  hydroxy-acids  (tyrosine,  serine  and  oxyproline)  form,  as  a 
rule,  only  a  small  proportion  of  the  products  of  protein  hydrolysis. 
Direct  union  of  carbon  atoms,  therefore,  cannot  be  a  frequent  mode  of 
linkage  of  amino-acids  within  the  protein  molecule. 

B.  Ether-like  unions,  as: 

I  I 

I  | 

Such  unions,  however,  would  only  be  possible  when  one  of  the  two 
amino-acids  thus  united  contained  a  hydroxyl-group  and,  as  we  have 
seen,  hydroxy-amino-acids  constitute  only  a  small  proportion  of  the 
total  amino-acids  yielded  by  a  protein  when  it  is  hydrolyzed. 

C.  The  carbon  atoms  may  be  united  through  a  nitrogen  atom  as: 

I      I      I 

— C— N— C— 

I  I 

Several  varieties  of  this  mode  of  union  are  possible,  as: 

— CH*— NH— CH2—  —  CH*— NH— CO— 

I.  II. 

CH2— N(OH)    =  C—  — CH2— NH— C(NH)— 

III.  IV. 


TYPES  OF  UNION  IN  THE  PROTEIN  MOLECULE  149 

In  the  synthetic  polypeptides  it  may  be  inferred  from  the  methods 
of  synthesis  employed  that  the  amino-acids  are  united  with  one  another 
through  a  nitrogen  atom,  and  the  fact  that  these  polypeptides  yield 
amino-acids  on  treatment  with  the  same  hydrolyzing  agents  that 
produce  amino-acids  from  proteins,  and  the  fact  that  polypeptides  in 
which  this  structure  has  been  established  by  synthesis,  occur  among 
the  partial  digestion-products  of  protein,  combine  to  establish  the 
correctness  of  the  view  that  in  the  proteins,  as  in  the  synthetic  poly- 
peptides, the  union  of  amino-acids  takes  place  through  the  neutraliza- 
tion of  amino-groups  by  carboxyl-groups.  Furthermore  the  fact  that 
the  proteins  yield  the  Biuret-reaction  also  confirms  this  view  of  the 
construction  of  the  protein  molecule.  It  has  been  found  that  only  those 
substances  which  contain  two — COHN — groups  or  two  — CSHN— 
or  two  — C(NH)HN —  groups,  or  under  certain  conditions,  two 
— CHNH —  groups  yield  the  biuret-test.  That  such  groups  as 


— COHN— c— COHN— 

I 

occur  frequently  in  protein  molecules,  as  they  do  in  the  molecules  of 
the  polypeptides,  is  therefore,  established  by  a  number  of  independent 
lines  of  evidence,  and  the  existence  of  a  large  number  of  such  linkages 
in  the  proteins  is  furthermore  confirmed  by  the  extreme  paucity  of 
Free  Amino-groups  in  the  native  protein  molecule. 

The  content  of  free  amino-groups  in  native  and  derived  proteins 
may  be  estimated  in  either  of  two  ways.  The  first  depends  upon  the 
liberation  of  nitrogen  which  occurs  when  free  — NH2  groups  react  with 
nitrous  acid,  in  accordance  with  the  equation: 

RNH2     +     HNO2     =     ROH     +    H2O     +     N2 

one  molecule  of  nitrogen  being  released  for  every  free  amino-group 
originally  present  in  the  substance.  The  second  method  (Sorensen's 
method  of  formol-titration)  depends  upon  the  fact  that  formaldehyde 
reacts  with  amino-acids  to  form  methylene  derivatives  in  accordance 
with  the  reaction: 

HOOCRNH2  +  HCOH  =  HOOCRN:CH2  +  H2O. 

If  the  solution  be  neutral  to  begin  with,  it  will  now  be  acid,  owing  to 
the  destruction  of  the  basic  — NH2-group  leaving  the  carboxyl-group 
unopposed.  The  number  of  amino-groups  destroyed  by  the  formalde- 
hyde, or  rather  the  number  of  carboxyl-groups  left  unopposed,  may  be 
estimated  by  the  quantity  of  alkali  required  to  restore  the  original 
neutral  reaction  of  the  solution. 

An  examination  of  the  various  proteins  by  either  of  these  methods 
reveals  the  fact  that  the  content  of  free  amino-groups  in  the  unhydro- 


150  COMPOUNDS  OF  THE  PROTEINS 

lyzed  protein  molecules  is  very  small  indeed.    Thus  Van  Slyke  and 
Birchard  have  obtained  the  following  results: 

PERCENTAGE  OF  TOTAL  NITROGEN  PRESENT  IN  FREE  AMINO 

GROUPS. 

Hemoglobin 6.0 

Casein 5.5 

Hemocyanin 4.3 

Gelatin 3.1 

Edestin 1.8 

Gliadin .  1.1 

Zein 0.0 

Heteroalbumose 8.1 

Protoalbumose 9.9 

On  the  other  hand,  Edestin,  after  complete  hydrolysis  by  hydrochloric 
acid,  yields  a  volume  of  free  nitrogen,  on  treatment  with  nitrous  acid, 
corresponding  to  no  less  than  seventy-nine  per  cent,  of  its  total  nitrogen 
content,  and  the  formol-titration  is  proportionately  increased.  A  very 
small  proportion  of  the  amino-groups  of  the  amino-acids  from  which 
these*  proteins  are  built  up  are,  therefore,  present  as  unneutralized 
amino-groups  in  the  unhydrolyzed  protein. 

It  has,  in  fact,  been  shown  that  the  free  amino-nitrogen  in  the 
unaltered  protein  molecule  exactly  corresponds  in  quantity  to  one-half 
the  Lysine  nitrogen.  Hence  Zein,  which  contains  no  lysine  radicals, 
yields  no  free  nitrogen  on  treatment  with  nitrous  acid.  The  period 
required  for  the  complete  interaction  of  proteins  with  nitrous  acid  is 
about  thirty  minutes  (with  the  technique  employed  by  Van  Slyke)  which 
is  ten  times  as  long  as  the  period  required  for  complete  interaction  with 
a-ami no-groups,  but  corresponds  exactly  with  that  found  to  be  required 
for  the  complete  interaction  of  nitrous  acid  with  the  co-ammo-group 
of  lysine.  From  these  facts  Van  Slyke  and  Birchard  infer  that  lysine 
is  united  to  the  adjacent  amino-acid  radicals  in  the  protein  molecule 
by  means  of  its  carboxyl-  and  its  a-amino-groups,  while  the  w-amino- 
group  remains  uncombined  and  represents,  within  at  most,  a  fraction 
of  a  per  cent,  of  the  total  protein  nitrogen,  the  entire  amount  of  free 
amino-nitrogen  in  the  native  proteins.  The  a-amino-groups,  which 
constitute  by  far  the  greater  part  of  the  free  amino-nitrogen  formed 
after  complete  hydrolysis  are,  in  the  intact  protein  molecule,  all  con- 
densed into  peptide-linkages. 

In  the  primary  Proteoses,  or  first  split-products  of  protein  hydrolysis, 
the  relations  are  different.  The  free  amino-groups  in  hetero-  or  proto- 
albumose  exceed  one-half  the  content  of  lysine  nitrogen  by  3.0  and  4.8 
per  cent,  of  the  total  nitrogen  respectively,  indicating  that  an  appreci- 
able proportion  of  the  a-amino-groups  are  uncovered  even  in  the  first 
steps  of  hydrolysis. 

Notwithstanding  the  fact,  however,  that  a  great  majority  of  the 
linkages  uniting  the  different  amino-acids  in  the  protein  molecule  have 
thus  been  proved  to  be  of  the  peptide  character,  the  possibility  is  by 


POLYPEPTIDE  STRUCTURE  OF  PROTEINS  151 

no  means  excluded  that  other  linkages  may  coexist  with  these  in  the 
protein  molecule.  One  fact  which  would  appear  to  afford  indication 
of  the  presence  of  non-peptide  linkages  in  the  native  protein  molecule 
is  that  while  native  protein  is  attacked  by  the  Pepsin  of  gastric  juice 
and  hydrolyzed  by  it  as  far  as  the  proteose  stage  of  hydrolysis,  the 
various  peptones  and  synthetic  polypeptides  are  not  hydrolyzable  by 
pepsin.  This  may  mean  that  types  of  union  exist  in  the  protein 
molecule  which  are  susceptible  to  attack  by  pepsin  and  which  are  not 
present  in  the  peptones  and  polypeptides.  On  the  other  hand  it  may 
simply  indicate  that  the  greater  length  of  the  amino-acid  chain  in  the 
native  protein  molecule  confers  upon  it  sufficient  instability  to  lay  it 
open  to  attack  and  disintegration  by  the  relatively  weak  hydrolyzing 
enzyme,  pepsin,  while  the  relatively  stable  proteoses  require  a  more 
energetic  hydrolyzing-agent  such  as  the  Trypsin  of  pancreatic  juice. 
This  latter  view  receives  substantial  support  from  the  fact  that  the 
splitting  of  protein  into  proteoses  already  results  in  the  uncovering 
of  free  amino-groups,  so  that  the  proteoses  are  evidently  united  together 
in  the  protein  molecule  through  nitrogen  atoms  as  the  amino-acids 
are  linked  together  in  peptides.  Furthermore,  it  is  definitely  known 
that  greater  length  of  an  amino-acid  chain  confers  upon  it  greater 
instability  toward  hydrolyzing  enzymes.  Thus,  Tetra-glycyl-glycine 
is  hydrolyzed  by  trypsin,  while  glycyl-glycin,  diglycyl-glycine  and 
triglycyl-glycine  are  not  attacked  by  this  enzyme. 

CONSEQUENCES  OF  THE  POLYPEPTIDE  STRUCTURE  OF 
PROTEINS. 

The  polypeptides  are  as  essentially  amino-acids  as  the  amino-acids 
out  of  which  they  are  built  up.  Thus,  glycyl-glycine  is  as  typically 
an  amino-acid  as  glycine  itself,  since  it  possesses  an  — NH2  group  as 
well  as  a  — COOH  group,  and  for  this  reason  is  presumably  capable  of 
forming  compounds,  not  only  with  acids  and  bases,  but  also,  possibly, 
even  with  neutral  salts,  by  attaching  the  basic  radical  of  the  salt  at 
one  point  of  the  molecule  (the  carboxyl)  and  the  acid  radical  at  another 
(the  amino-group) .  On  undergoing  electrolytic  dissociation  in  aqueous 
solution  it  may  be  supposed  to  yield  either  hydrogen  (H+)  ions,  or 
hydroxyl  (OH~)  ions,  owing  to  the  occurrence  of  a  reaction  with  water 
of  the  type: 

/NH2  /NH3OH 

R<        +  H.OH  =  R< 

XCOOH  XCOOH 

just  as  ammonia,  in  aqueous  solution,  partially  reacts  with  water  to 
form  NH4  OH. 

It  was  considered  until  quite  recently,  and  is  still  thought  in  some 
quarters,  that  these  elements  in  the  structure  of  the  protein  and 
polypeptide  molecules  afford  an  explanation  of  the  power  which  they 
possess  of  uniting  with  both  acids  and  bases,  in  other  words  the 


152  COMPOUNDS  OF  THE  PROTEINS 

Amphoteric  character  of  the  proteins.  A  variety  of  facts  have  been 
ascertained  in  recent  years,  however,  which  have  compelled  a  revision 
of  this  opinion,  and  we  now  recognize  that  some  elements  in  the  protein 
molecule  other  than  free  — NH2  or  COOH  groups  must  be  responsible 
for  the  acid-  and  base-neutralizing  power  that  is  possessed  in  a  marked 
degree  by  many  proteins. 

In  the  first  place,  the  investigations  to  which  reference  has  been 
made  above  have  shown  that  only  a  very  small  proportion  of  the 
nitrogen  in  proteins  is  present  within  their  molecules  in  the  form  of 
— NH2  groups.  Thus,  in  the  case  of  Edestin,  as  the  above-quoted 
estimations  show,  only  1.8  per  cent,  of  the  total  nitrogen  is  present  in 
the  form  of  — NH2  groups.  Now  edestin  is  insoluble,  when  in  the  free 
condition,  in  water.  It  forms  an  insoluble  hydrochloride  containing 
14X10"5  equivalents1  of  hydrochloric  acid  per  gram.  On  further 
addition  of  acid,  soluble  hydrochlorides  are  formed,  and  the  substance 
passes  completely  into  solution  when  the  proportion  of  combined  acid 
is  just  double  that  contained  in  the  insoluble  hydrochloride.  Still 
further  additions  of  acid,  however,  continue  to  be  neutralized  by  the 
protein  until,  at  neutrality  to  tropaeolin  OO,  which  changes  color  when 
the  amount  of  free  acid  in  solution  is  between  one-hundredth  and  one- 
thousandth  normal,  after  due  allowance  for  the  acid  which  remains 
unneutralized,  it  is  found  that  edestin  combines  with  no  less  than 
127  X  10~5  equivalents  of  acid  per  gram.  The  formation  of  the  insoluble 
compound  with  14XlO~5  equivalents  of  acid  must  correspond  to  the 
union  of  at  least  one  molecule  of  the  acid  with  each  molecule  of  protein, 
for  any  acid  in  excess  of  this  amount  results  in  the  formation  of  a 
compound  of  quite  a  different  character,  namely,  one  which  is  soluble 
in  water.  The  maximal  number  of  molecules  of  acid  which  may  be 
neutralized  by  a  molecule  of  edestin  has  not  been  determined,  but  it 
evidently  cannot  be  less  than  the  number  corresponding  to  127X10"5 
equivalents  of  hydrochloric  acid  per  gram  of  protein.  If  we  assume, 
therefore,  that  the  insoluble  compound  represented  the  result  of  union 
of  one  molecule  of  acid  with  each  molecule  of  protein,  then  the  com- 
pound formed  at  neutrality  to  tropaeolin  must  represent  the  formation 

of  a  compound  containing  -T-T-  =  9  molecules  of  acid  for  each  molecule  of 

edestin.  If  this  compound  were  formed  by  the  union  of  the  acid  with 
— NH2  groups  in  accordance  with  a  series  of  reactions  of  the  type : 

RNHsOH     +     HCl      =     RNH2HC1     +     H2O 

then  we  would  obviously  have  to  assume  the  existence  of  no  fewer  than 
nine  free  — NH2  groups  in  the  molecule  of  edestin.  If  we  assume  the 
insoluble  compound  to  have  been  formed  by  the  union  of  two  molecules 
of  acid  with  one  of  edestin,  then  the  estimate  of  the  number  of  free 
— NH2  groups  must  be  raised  to  2X9=  18  and  so  forth. 

1  That  is,  the  combining  weight  of  HCl  expressed  in  grams  and  multipled  by  0.00014, 
or  the  hydrochloric  acid  which  is  present  in  1.4  c.c.  of  a  tenth  normal  solution. 


POLYPEPTIDE  STRUCTURE  OF  PROTEINS  153 

Supposing  the  insoluble  compound  to  have  been  formed  by  the  union 
of  only  one  molecule  of  acid  with  one  molecule  of  edestin,  since  1  gram 
of  the  protein  is,  in  this  compound,  equivalent  to  only  14X10"5  mole- 
cule of  hydrochloric  acid,  about  7000  grams  of  protein  would  be 
neutralized  by  one  gram-molecule  of  hydrochloric  acid.  The  molecular 
weight  of  edestin,  if  the  insoluble  compound  is  formed  by  the  union  of 
one  molecule  of  hydrochloric  acid  with  one  molecule  of  protein,  must 
therefore,  be  about  7000.  Nine  — NH2  groups  in  this  molecule  would 
correspond  to  ten  per  cent,  of  the  total  which  edestin  contains.  If  we 
were  to  assume  that  the  insoluble  compound  contains  two  molecules  of 
hydrochloric  acid  per  molecule  of  protein,  then  our  estimate  of  the 
molecular  weight  of  edestin  would  have  to  be  doubled,  but  as  the 
estimated  number  of  free  — NH2  groups  would  also  be  doubled,  this 
would  leave  us  still  the  same  proportion,  ten  per  cent.,  of  the  total 
nitrogen  in  the  molecule  in  the  form  of  free  — NH2  groups. 

Now  the  measurements  of  Van  Slyke  and  Birchard  have  shown  that 
only  1.8  per  cent,  of  the  total  nitrogen  of  the  edestin  molecule  is 
present  therein,  in  the  form  of  free  — NH2,  groups,  so  that  no  less  than 
8.2  tenths  or  eighty-two  per  cent,  of  the  neutralizing-power  of  edestin 
for  acids  remains  to  be  accounted  for  in  some  other  fashion  than  by 
the  assumption  of  a  union  of  the  acid  with  free  — NH2  groups. 

The  same  or  similar  measurements  have  been  made,  employing  a 
diversity  of  proteins,  arid  always  with  the  same  disparity  between  the 
actual  proportion  of  free  amind-groups  in  the  molecule  of  the  protein 
and  the  proportion  which  would  be  required  to  accomplish  the  neutral- 
ization of  all  the  acid  which  the  protein  is  capable  of  binding.  Thus, 
it  has  been  pointed  out  by  Kossel  and  Cameron  that  the  acid-combining 
capacity  of  the  protamine,  Salmine  is  equal  to  the  combining-capacity 
of  all  of  the  guanidine-groups  of  the  arginine  radicals  which  this  protein 
contains.  Yet  salmine  yields  no  nitrogen  whatever  on  treatment  with 
nitrous  acid.  Sturine,  which  is  another  protamine,  contains  67  per  cent, 
of  its  nitrogen  in  the^form  of  histidine  and  6  to  7  percent,  in  the  form 
of  lysine.  It  yields  nitrogen  on  treatment  with  nitrous  acid  corre- 
sponding to  the  co-amino-group  of  the  lysine.  Only  about  three  or  four 
out  of  every  hundred  nitrogen  atoms  in  sturine  are  therefore  present 
in  the  form  of  free  amino-groups.  Yet  one  hundred  nitrogen  atoms  in 
sturine  will  neutralize  no  less  than  twenty-four  equivalent  gram-mole- 
cules of  acid.  Evidently  at  least  twenty  of  these  acid  molecules  must 
attach  themselves  to  the  molecule  of  protein  at  some  other  points  than 
those  provided  by  free  — NH2-groups. 

The  number  of  Free  Carboxyl-groups  in  any  protein  cannot  be  much 
in  excess  of  the  number  of  free  amino-groups,  for  otherwise  the  protein 
would  be  overwhelmingly  acid  in  its  character,  and  behavior,  and 
besides,  since  relatively  few  of  the  amino-acid  radicals  in  most  proteins 
are  dicarboxylic  acid  radicals,  if  a  great  excess  of  free  carboxyl-groups 
were  present  in  the  molecule,  the  combined  amino-groups  could  not 
all  be  attached  to  carboxyl-groups  as  they  are  in  the  polypeptides,  and 


154  COMPOUNDS  of  THE  PROTEINS 

as  all  our  evidence  tends  to  show  they  are  in  the  proteins.  Moreover 
the  results  of  the  formol-titration  show  that  there  are  not  many  free 
carboxyl-groups  in  the  protein  molecule,  and  the  same  conclusion  may 
be  reached  from  a  consideration  of  the  effects  of  rather  concentrated 
alkali  in  bringing  about  "  racemization"  or  optical  inactivity  of  the 
majority  of  the  amino-acid  radicals  in  proteins.  It  is  found  that  amino- 
acid  radicals  of  which  the  carboyxl-group  remains  uncombined,  are  not 
"racemized"  by  alkali,  while  amino-acids  of  which  the  carboxyl-groups 
are  neutralized  in  peptide-linkages  are  rendered  optically  inactive  by 
strong  alkalies.  The  great  majority  of  the  amino-acids  which  result 
from  the  alkaline  hydrolysis  of  proteins  are  optically  inactive  and  so  we 
must  assume  that  in  the  native  protein  molecule  their  carboxyl-groups 
were  not  unattached. 

Now  uncombined  Casein  is  insoluble  in  water,  but  when  combined 
with  acids  or  with  bases  it  is  soluble.  When  just  sufficient  alkali  has 
been  employed  to  carry  every  particle  of  casein  into  solution  at  least 
one  molecule  of  the  alkali  must  have  combined  with  each  molecule  of 
casein.  To  carry  one  gram  of  casein  into  solution  11.4X10"5  equiva- 
lents of  base,  or  1.14  c.c.  of  tenth  normal  alkali  just  suffice,  indicating 
a  combining-weight  for  casein  of  about  8800.  The  ty rosin  and 
sulphur-contents  of  casein  indicate  that  the  molecular  weight  of  casein 
must  be  some  multiple  of  4400. 

In  the  presence  of  excess  of  alkali,  however,  the  combining  capacity 
of  casein  for  bases  is  very  much  greater.  We  cannot,  of  course,  deter- 
mine the  maximal  combining-capacity  of  casein  for  bases  by  titration, 
because  the  removal  of  the  uncombined  excess  of  alkali  by  the  acid 
used  in  titration  simply  results  in  reducing  the  combining-capacity  of 
the  casein  for  the  alkali,  just  as  the  running  in  of  acid  into  a  solution 
of  sodium  carbonate  results  in  the  formation  of  sodium  bicarbonate. 
Nor  is  it  convenient  to  determine  the  maximal  combining-capacity 
of  proteins  for  bases  by  means  of  indicators.  The  method  employed 
is  to  determine  the  quantity  of  uncombined  alkali  in  the  protein 
solution  electrometrically  by  means  of  the  Gas-chain  (see  Chapter  XII), 
that  is  by  the  potential  developed  at  the  surface-layer  of  an  electrode 
of  hydrogen  dipped  into  the  protein  solution.  The  greater  the  con- 
centration of  free  alkali,  i.  e.,  of  hydroxyl  ions  in  the  solution  the  less, 
in  proportion,  must  be  the  concentration  of  free  hydrogen  ions,  and 
the  less  the  concentration  of  free  hydrogen  ions  in  the  solution  the  more 
hydrogen  ions  will  travel  from  the  superficial  layer  of  the  electrode 
into  the  superficial  layer  of  solution  which  is  in  contact  with  it.  These 
hydrogen  ions  carry  with  them  a  positive  charge,  and  hence  the  solution 
becomes  charged  positively  and  the  electrode  carries  a  corresponding 
negative  charge.  The  potential  thus  created  is  a  measure  of  the 
alkalinity  (or  acidity)  of  the  solution  under  investigation.  For  the 
Hydrogen  Electrode  we  use  a  piece  of  platinum  foil  or  platinum  gauze 
coated  with  platinum-black  and  saturated  with  hydrogen  gas. 


POLYPEPTIDE  STRUCTURE  OF  PROTEINS  155 

By  this  method  it  may  be  shown  that  in  the  presence  of  an  excess 
of  alkali,  casein  combines  with  a  maximal  proportion  of  180X10"5 
equivalents  of  base  per  gram.  The  combining-capacity  of  casein  for 
alkalies  does  not  exceed  this  figure  no  matter  what  excess  of  alkali 
we  may  employ.  We  have  seen  that  the  minimal  combining-capacity 
of  casein  for  bases  is  11.4  equivalents  of  base  per  gram.  Hence,  reason- 
ing as  we  did  in  the  case  of  the  compounds  of  edestin  with  hydrochloric 
acid,  if  the  minimal  proportion  of  alkali  which  just  suffices  to  carry 
casein  into  solution  corresponds  to  the  union  of  one  molecule  of  alkali 
with  one  molecule  of  casein,  the  maximal  proportion  of  alkali  which 
may  be  bound  by  casein  must  correspond  to  the  union  of  at  least  sixteen 
molecules  of  base  with  one  molecule  of  protein.  If  these  molecules  of 
alkali  were  united  to  the  protein  through  — COOH-groups  there  must 
be  sixteen  of  them,  or  over  one-fourth  of  all  of  the  carboxyl-groups  in 
the  protein  must  exist  therein  in  the  free,  uncombined  condition.  We 
have  seen  that  this  would  be  impossible  excepting  in  the  case  of  the 
second  carboxyl  in  the  dicarboxylic  acid  radicals  and  of  these  there  are 
only  sufficient  in  casein  to  supply  one-half  of  the  carboxyl-groups 
required.  Evidently  the  union  of  bases  with  casein  is  accomplished 
through  some  agency  other  than  free  carboxyl-groups. 

The  nature  of  the  radicals  which  accomplish  the  union  of  protein 
with  alkalies  is  indicated  by  the  experiments  of  H.  M.  Vernon,  who  has 
compared  the  power  of  proteins  and  of  their  hydrolytic  decomposition 
products  to  neutralize  bases.  Although  the  Hydrolytic  Decomposition- 
products  of  a  protein  will  neutralize  more  alkali  than  the  undecomposed 
protein,  yet  the  gain  in  power  to  neutralize  bases  is  very  much  less 
than  one  would  anticipate  in  view  of  the  large  number  of  carboxyl- 
groups  which  are  set  free  by  hydrolysis.  In  fact  the  alkali-neutralizing 
power  of  the  hydrolyzed  protein  is  only  slightly  greater  than  the 
alkali-neutralizing  power  of  the  native,  undecomposed  protein.  Now 
in  the  process  of  hydrolysis  the  — COHN —  groups  of  the  protein  are 
split  into  — NH2  and  — COOH  groups.  The  inference  is  that  the 

—  COHN groups  within  the  protein  molecule  must  be  nearly  as 

efficient  in  accomplishing  the  neutralization  of  bases  as  the  — COOH 
groups  of  the  constituent  amino-acids  out  of  which  the  protein  is 
built  up. 

More  direct  evidence  that  the  —COHN —  groups  in  the  protein  mole- 
cule are  responsible  for  the  neutralization  of  bases  by  proteins  is  afforded 
by  the  investigations  of  Osborne  and  Leavenworth,  who  have  shown 
that  Edestin,  for  example,  combines  with  and  holds  in  solution  34.67 
per  cent,  of  its  weight  of  copper  in  the  form  of  the  otherwise  insoluble 
cupric  hydroxide.  This,  if  we  assume  that  each  copper  atom  unites 
with  one  nitrogen  atom,  involves  the  union  of  cupric  hydroxide  with 
ten  out  of  every  sixteen  atoms  of  nitrogen  in  the  edestin  molecule. 
Now  this  is  exactly  the  proportion  of  nitrogen  which  edestin  yields  in 
the  form  of  amino-nitrogen  after  complete  hydrolysis.  In  other  words, 


156  COMPOUNDS  OF  THE  PROTEINS 

it  is  exactly  equal  to  the  proportion  of  — COHN groups  which  the 

unhydrolyzed  molecule  contains;  precisely  similar  results  were  obtained 
with  Gliadin. 

Direct  proof  on  the  other  hand,  that  free  — NH2-groups  are  not 
responsible  for  any  appreciable  proportion  of  the  acid-combining 
capacity  of  proteins  has  been  furnished  by  the  experiments  of  Blasel 
and  Matula,  and  of  Pauli  and  Hirschfeld.  These  investigators  prepared 
Deaminized  Gelatin  by  acting  upon  gelatin  with  nitrous  acid,  thus 
destroying  all  the  free  — NH2-groups  in  the  molecule.  They  then 
compared,  with  the  aid  of  the  hydrogen  electrode,  the  acid-combining 
capacity  of  the  deaminized  gelatin  with  that  of  normal  gelatin.  They 
found  that  the  combining-capacity  of  deaminized  gelatin  for  acids  is 
only  very  slightly  inferior  to  that  of  normal  gelatin,  indicating,  beyond 
any  question,  that  the  combining-capacity  of  gelatin  for  acids  is,  in 
very  large  proportion,  attributable  to  elements  of  the  molecule  other 
than  free  — NH2-groups.  Since  nitrogen  atoms  must  certainly  be  the 
agents  through  which  union  of  acids  with  protein  is  brought  about, 
the  inference  is  unavoidable  that  the  elements  of  the  molecule  which 
actually  accomplish  the  binding  of  acids  by  protein  are,  in  very  large 
proportion  the  — COHN — -groups  within  the  body  of  the  protein 
molecule. 

To  account  for  both  the  acid-  and  the  base-combining  capacity  of 
the  proteins  we  must  therefore  look,  not  to  the  small  proportion  of 
free  — NH2  or  — COOH-groups  which  the  proteins  afford,  but  to  the 

— COHN groups  within  the  body  of  the  molecule.  Now  two  varieties 

of  this  linkage  can  be,  conceived,  between  which  it  has  not  proved 
possible  as  yet,  to  decide  by  any  direct  method  of  analysis.  Thus 
Glycyl-glycine  may  conceivably  be  either: 

Keto-Form. 
H2N.CH2.CO— HN.CH2.COOH 

or: 

Enol-Form. 
H2N.CH2.C(OH)  =N.CH2.COOH 

and  our  analytical  data,  and  the  modes  of  decomposition  and  synthesis 
of  the  proteins  and  peptides  do  not  enable  us,  with  any  degree  of  cer- 
tainty, to  distinguish  between  them.  Neither  form  is  therefore  incon- 
sistent with  our  present  knowledge  of  the  synthesis  and  hydrolysis  of 
proteins  and  polypeptides,  but  while  the  keto-form  of  the  — COHN— 
-group  would  conceivably  possess  the  power  of  neutralizing  acids,  it 
offers  no  probable  point  of  union  for  bases.  The  enol-form,  on  the 
contrary,  would  provide  a  point  of  union  for  either  acids  or  bases. 

According  to  Werner's  theory  of  valencies,  the  nitrogen  in  either  of 
these  types  of  linkage  contains  two  latent  valencies,  positive  and 
negative,  which,  while  the  nitrogen  remains  trivalent,  neutralize  one 
another,  but  when  the  nitrogen  becomes  pentavalent  are  capable, 
respectively,  of  neutralizing  a  negative  and  a  positive  radical.  The 


POLYPEPTIDE  STRUCTURE  OF  PROTEINS 


157 


enol  type  of  union  carries  with  it  the  possibility  of  the  following  types 
of  reaction : 

H 


—  COH=N— 


Na 


OH" 


and 


—  COH—  N—     +     H+     + 


(—  CONa)++ 


(—  COH) 


yielding,  in  each  case  only  protein  ions. 

This  conception  of  the  mode  of  union  of  proteins  with  inorganic 
acids  and  bases  affords  an  explanation  of  what  would  otherwise  con- 
stitute a  very  puzzling  fact,  namely,  that  while  compounds  of  acids 
or  bases  with  protein  form  very  good  conductors  of  electricity  through 
their  solutions,  and  must,  therefore,  be  electrolytically  dissociated  into 
ions,  yet  no  evidence  is  afforded  by  these  solutions  of  the  existence  in 
them  of  any  ions  derived  from  the  combined  acid  or  base.  Thus 
chlorides  of  the  proteins  yield  only  traces  of  chlorine  ions  in  solution, 
as  is  shown  by  the  difficulty  or  sluggishness  with  which  they  react  with 
silver  salts  to  form  silver  chloride.  The  calcium  compound  of  Casein 
does  not  dissociate  any  appreciable  proportion  of  calcium  ions,  and 
compound  of  the  proteins  with  silver,  mercury,  lead,  copper,  etc.,  do 
not  yield  up  these  ions  to  the  solution.  The  conductance  of  electricity 
through  the  solution  of  a  protein  compound  cannot  be  due  to  contami- 
nation of  the  protein  with  dissociable  inorganic  salts,  because  the  con- 
ductivities observed  are  too  large  to  be  accounted  for  in  this  way  and, 
moreover,  the  conductivity  of  the  protein  compound  varies  in  a  very 
striking  and  regular  manner  with  the  proportion  of  acid  or  alkali 
bound,  increasing,  as  one  might  expect,  with  the  number  of  acid  or 
alkali  radicals  which  have  entered  into  combination  with  the  protein. 
Furthermore,  in  many  instances,  e.  g.,  Casein  and  Serum-globulin,  it  is 
easy  to  show  that  the  protein  participates  in  the  conductance  of  the 
current  owing  to  the  fact  that  the  protein  is  precipitated  at  one  of 
the  electrodes,  and  the  amount  of  protein  so  precipitated  is  proportional 
to  the  amount  of  current  which  has  traversed  the  solution.  It  is  a 
very  striking  fact  that  casein  in  alkaline  solution,  although  it  is  only 
precipitated  at  the  one  electrode  (the  anode)  yet  migrates  toward  both 
electrodes  when  a  current  is  passed  through  the  solution,  indicating 
that  certain  portions  of  the  casein  are  engaged  in  transporting  positive, 
while  others  are  transporting  negative  charges.  Migration  of  the 
proteins  toward  both  electrodes  has  also  been  observed  in  solutions  of 
Hemoglobin  and  in  solutions  of  Fibrinogen. 


158  COMPOUNDS  OF  THE  PROTEINS 

THE  PRECIPITATION  AND  COAGULATION  OF  PROTEINS 
BY  INORGANIC  SALTS. 

The  precipitation  of  proteins,  and,  indeed,  of  Colloids  in  general, 
may  be  of  two  kinds :  The  first  is  clearly  accompanied  by  decomposi- 
tion of  the  precipitating  agent,  it  will  not  occur  unless  the  protein  is 
ionized,  i.  e.,  migrates  under  the  influence  of  an  electric  current;  and 
only  small  quantities  of  the  precipitating-agent  are  required  to  bring 
about  the  precipitation.  The  second  kind  of  precipitation,  however, 
whether  accompanied  by  decomposition  of  the  precipitating-agent  or 
not,  occurs  even  when  the  protein  is  non-ionized,  and  requires  relatively 
large  amounts  of  the  precipitating-agent.  Precipitation  of  the  first 
kind  is,  generally  speaking,  only  brought  about  by  electrolytes,  while 
precipitation  of  the  second  kind,  although  as  a  rule,  more  readily 
brought  about  by  electrolytes  than  by  non-electrolytes,  may  neverthe- 
less be  brought  about  by  certain  non-electrolytes,  for  example,  by 
alcohol.  For  this  latter  type  of  precipitation  we  shall  henceforth 
reserve  the  term  Coagulation. 

Both  Precipitation  and  Coagulation  of  a  protein  may  be  brought  about 
by  one  and  the  same  inorganic  salt.  In  such  a  case  the  gradual  addi- 
tion of  salt  to  the  originally  salt-free  solution  which  contains  ionic 
protein,  i.e.,  protein  which  drifts  to  one  electrode  or  to  the  other  in  an 
electric  field,  first  brings  about  precipitation  and  then  resolution  of  the 
protein.  In  this  new  solution  the  protein  appears  to  be  invariably 
non-ionic,  and  it  can  be  coagulated  by  still  further  addition  of  the  salt. 

The  first  kind  of  precipitation  appears  to  be  undoubtedly  chemical 
in  character  and  in  mechanism.  The  mechanism  of  coagulation  is, 
however,  far  from  clear,  and  for  the  attainment  of  an  adequate  under- 
standing of  this  phenomenon  we  shall  doubtless  have  to  wait  until 
the  physicochemical  theory  of  phenomena  of  solution  in  general  has 
reached  a  more  mature  stage  of  development  than  it  has  at  present. 
There  would  appear  to  be  no  room  for  doubt,  however,  that  processes 
of  Dehydration  play  an  important  and  perhaps  a  decisive  part  in  bringing 
about  coagulation. 

The  concentrations  of  the  various  inorganic  salts  which  are  required 
to  bring  about  the  precipitation  of  a  colloid  in  solution  very  greatly 
depend  upon  the  Electrical  Sign  of  the  inorganic  ions  with  which  it  may 
chance  to  be  combined,  and  upon  the  Valencies  of  the  ions  of  the  salts 
used  for  precipitating  the  colloid.  Thus,  defining  the  "  Precipitating- 
power"  of  a  salt  as  the  reciprocal  of  the  concentration,  in  gram-molecules 
per  liter,  necessary  to  precipitate  a  given  solution  of  colloidal  Sulphide 
of  Arsenic,  Schultz  found  that  the  relative  precipitating-powers  of  the 
univalent,  divalent  and  trivalent  metals  are  in  the  ratios  1  :  30  :  1650. 
Similar  figures  were  obtained  for  colloidal  Cadmium  Sulphide,  while 
Linder  and  Picton,  using  colloidal  Antimony  Sulphide,  found  that  the 
precipitating-powers  of  different  salts  of  a  given  metal  are  proportional 
to  their  equivalent  conductivities,  i.  e.,  to  the  concentration  of  metal 


PRECIPITATION  OF  PROTEINS  BY  INORGANIC  SALTS          159 

ions  present  in  the  mixture,  and  that  the  relative  precipitating-powers 
of  the  sulphates  of  univalent,  divalent  and  trivalent  metals  can  be 
expressed  by  the  ratios  1 :  35  :  1923. 

In  all  of  these  cases  the  colloid  employed  was  Electronegative ;  that  is, 
in  electrolysis  it  was  precipitated  at  the  anode,  the  colloid  behaving 
like  the  acid  radical  of  a  salt.  The  experiments  which  we  have  cited 
show  that  in  such  instances  the  ion  of  the  added  electrolyte  which  is 
effective  in  bringing  about  precipitation  is  the  cation,  since  the  valency 
of  the  cation  determined  the  precipitating-power  of  the  salt,  while  the 
valency  of  the  anion  was  immaterial  to  the  result.  If,  however,  we 
dilute  the  white  of  a  a  egg  with  ten  times  its  volume  of  distilled  water, 
filter  off  the  flakes  of  Globulin  which  are  precipitated,  and  then  heat  the 
solution  to  boiling-point,  we  obtain  an  opalescent  solution  or  suspension 
of  Egg-albumin  which  is  very  easily  coagulated  by  traces  of  various 
salts.  If  a  trace  of  acid  be  added  to.  the  solution  and  an  electrical 
current  passed  through  it,  the  protein  is  precipitated  at  the  cathode, 
while  if,  instead  of  acid,  a  trace  of  alkali  be  added,  the  protein  is 
precipitated,  not  at  the  cathode  but  at  the  anode.  In  the  former  case 
the  protein  behaves  like  a  cation  or  the  basic  radical  of  a  salt,  and  is 
said  to  be  "Electropositive,"  while  in  the  latter  case  the  protein  behaves 
like  an  anion,  or  the  acid  radical  of  a  salt,  and  it  is  said  to  be  "Electro- 
negative." It  is  probable  that  in  both  these  instances  the  protein 
migrates  to  both  electrodes,  but  the  protein  ion  which  carries  the 
inorganic  radical  with  it,  in  the  first  case  the  acid  and  in  the  second  the 
alkali,  is  held  in  solution  by  the  acid  or  alkali  it  bears  after  it  has  given 
up  its  electrical  charge  to  the  electrode. 

Now  in  alkaline  solutions  of  this  heat-modified  egg-albumin  it  is 
found  that  the  cations  of  added  salts  are  the  active  agents  in  precipitat- 
ing the  protein,  just  as  in  the  case  of  the  sulphides  of  antimony,  arsenic 
or  cadmium,  but  in  acid  solutions  of  the  heat-modified  egg-albumin 
these  relationships  are  entirely  reversed,  the  valency  of  the  cation  of 
the  added  salt  becomes  immaterial  and  the  precipitating-power  of  the 
salt  is  determined  by  its  anion.  The  following  results,  obtained  by 
W.  B.  Hardy,  illustrate  this  inversion  of  the  precipitating  ion  when  the 
protein,  from  functioning  as  an  acid,  comes  to  function  as  a  base: 

PROTEIN  IN  PRESENCE  OF  A  TRACE  OF  ALKALI;  ELECTRONEGATIVE. 
Temperature  16  degrees.     Coagulating  salt  1  gram-mol.  in  80.000  c.c. 

Coagulated  at  once.  On  slightly  warming.  Did  not  coagulate. 
A12(SO4)3                            MgSO4  Na2SO4 

Cd(NO3)2  BaCl2  K2SO4 

CuSO4  CaCl2  NaCl 

CuCl2 

PROTEIN  IN  PRESENCE  OF  A  TRACE  OF  ACID;  ELECTROPOSITIVE. 

Coagulated  instantly.  No  effect. 
A12(SO4)3  CuCl2 

CaSO4  Cd(NO3)2 

K2SO4  BaCl2 

Na2SO4  NaCl 

MgSO4 


160  COMPOUNDS  OF  THE  PROTEINS 

Similar  results  have  been  obtained  with  a  variety  of  other  colloids, 
Electronegative  colloids  are  precipitated,  if  at  all,  by  cations;  electro- 
positive colloids  by  anions. 

Whetham  explained  these  phenomena  in  the  following  way:  He 
assumes  that  each  colloidal  particle  carries  ah  electrical  charge,  a 
corresponding  and  opposite  charge  being  induced  upon  the  surface  of 
the  water  in  immediate  contact  with  the  colloid.  The  effect  of  this 
charge  is  to  diminish  the  Surface-tension  at  the  surface  separating 
the  water  and  the  colloid,  and  therefore,  to  diminish  the  tendency  of 
this  surface  to  contract.  So  long  as  the  colloid  is  dispersed  through 
the  solution  in  the  form  of  minute  suspended  or  dissolved  particles 
the  surface  separating  the  colloid  and  the  water  is  very  large.  When 
the  colloid  is  flocculated  and  precipitated  the  surface  is,  in  consequence, 
contracted.  The  less  the  tendency  of  this  surface  to  contract, 
Whetham  argues,  the  less  will  be  the  tendency  for  the  colloidal  particles 
to  adhere  to  one  another  and  form  large  flocculi. 

The  cations  of  the  added  electrolyte,  in  the  case  of  "electronegative" 
colloids,  or  the  anions  of  the  added  electrolyte,  in  the  case  of  "  electro- 
positive" colloids,  neutralize,  according  to  Whetham,  the  charges  which 
are  carried  by  the  colloidal  particles.  The  electrical  double  layer  at 
the  surface  of  the  colloid  and  the  water  thus  disappears,  and  the  surface 
contracts;  the  finely  suspended  colloidal  particles  unite  to  form  large 
aggregates  having  a  less  extended  surface  and  these  aggregates  finally 
become  so  large  as  to  assume  the  properties  of  matter  in  mass,  and 
hence  are  carried  out  of  solution  by  the  action  of  gravity.  In  this 
way  the  dependence  of  the  precipitat ing-power  of  an  electrolyte  upon 
its  degree  of  ionization,  and  also  the  reversal  in  the  relative  precipitat- 
ing-powers  of  the  ions  of  the  added  electrolyte  upon  reversion  of  the 
sign  of  the  electrical  charge  presumed  to  be  carried  by  the  colloid, 
found  an  explanation.  In  interpreting  the  Valency  Rule  discovered 
by  Schultz,  Whetham  develops  his  theory  as  follows : 

"  In  a  solution  where  ions  are  moving  freely,  the  probability  that  an 
ion  is  at  any  instant  within  reach  of  a  fixed  point  is,  putting  certainty 
equal  to  unity,  approximately  represented  by  a  fraction  proportional 
to  the  ratio  between  the  volume  occupied  by  the  spheres  of  influence 
of  the  ions  and  the  whole  volume  of  the  solution  and  may  be  written 
as  A  C,  where  A  is  constant  and  C  represents  the  concentration  of  the 
solution.  The  chance  that  two  such  ions  should  be  present  together 
is  the  product  of  their  separate  chances,  that  is  (AC)2.  Similarly  the 
chance  for  the  conjunction  of  three  ions  is  (AC)3,  and  for  the  con- 
junction of  n  ions  is  (AC)n." 

"In  order  that  three  solutions  containing  trivalent,  divalent  and 
univalent  ions  respectively  should  have  equal  coagulative  powers, 
the  frequency  with  which  the  necessary  conjunctions  should  occur  must 
be  the  same  in  each  solution.  We  should  then  have,  the  constant  being 
assumed  equal  in  each  case: 

.  2n~  2n  3n~  3n  .  6n~  6n 

A    Ca          =     A    C2          =      A    Ci         —     a  constant     =     B 


PRECIPITATION  OF  PROTEINS  BY  INORGANIC  SALTS          161 

Therefore 

c,    -   ?*;  c2    =    ?*;  Cl    =    ^ 

A  A  A 

Ci,  €2,  C3  representing*  the  concentrations  of  monads,  diads  and  triads 
in  their  respective  solutions.  Thus  we  get  for  the  ratios  of  the  concen- 
trations of  equicoagulative  solutions: 

JL         i         -i  i-         -i 

Ci    :     C2    :     C3     =    B6n    :     B3n    :    B2n     =     1    :    B^n    :    B3n 

_L  ]_ 

Let  us  put  B6n    =  -  ;  the  ratios  can  then  be  written:    • 


The  reciprocals  of  the  numbers  expressing  the  relative  concentrations 
of  equicoagulative  solutions  give  values  proportional  to  the  coagulative 
powers  of  solutions  of  equal  concentration;  so  that,  calling  the  coagula- 
tive-powers  of  equivalent  solutions  containing  monovalent,  divalent 
and  trivalent  ions  respectively  px  :  p2  :  ps,  we  get: 

PI    :    p2    :    pa    :     =    1    :    x    :    x2 

Let  us  now  take  some  numerical  examples; 
Putting  x  =  32  we  get  the  series : 

l    :    32    :    1024 

which  agrees  very  well  with  Linder  and  Picton's  results  for  colloidal 
solutions  of  antimony  sulphide : 

l    :    35    :    1023 
and  putting  x=40,  we  get 

1    :    40    :     1600 

numbers  comparable  to  Schultze's  values  for  sulphide  of  arsenic/ 

This  theory  obtained  for  some  time  a  very  wide  acceptance,  but 
the  difficulty  attaching  to  it  from  the  first  was  that  of  accounting  for 
the  acquirement,  by  the  colloid,  of  an  electrical  charge.  It  will  be 
recollected  that  the  sign  of  the  charge  borne  by  the  portion  o*f  the 
colloid  which  is  precipitated  at  an  electrode  by  an  electric  current,  is 
reversed  by  changing  the  reaction  of  the  solution  of  the  colloid  from 
acid  to  alkaline.  Obviously,  therefore,  the  electrical  charge  carried 
by  the  colloidal  particles  is  determined  by  the  acid  or  alkali  which  is 
added  to  the  solution,  and  it  was  tacitly  assumed  that  the  charge 
obtaining  in  acid  solutions  was  derived  from  the  hydrogen  ions  of  the 
acid,  while  that  obtaining  in  alkaline  solutions  was  derived  from  the 
hydroxyl  ions  of  the  alkali.  But  if,  for  example,  egg-albumin  acquires 
11 


162  COMPOUNDS  OF  THE  PROTEINS 

a  positive  charge  from  the  hydrogen  ions  of  hydrochloric  acid,  we  must 
next  inquire  what  becomes  of  the  chlorine  ion  of  the  acid?  It  cannot 
exist  in  a  free  state  with  unneutralized  charges,  and  no  way  exists 
for  it  to  neutralize  its  charge  except  by  attachment  to  an  oppositely 
charged  albumin  molecule.  But  this  attachment  of  both  ions  of  the 
hydrochloric  acid  to  the  albumin  differs  in  no  distinguishable  way 
whatever  from  chemical  combination. 

That  the  hydrogen  ions  of  acids  are  actually  bound  by  the  protein 
may  readily  be  shown  by  employing  the  hydrogen  electrode  or  even 
by  the  aid  of  indicators.  The  anions  of  acids  must  therefore  be  like- 
wise attached  to  the  protein  particles.  Now  let  us  suppose  that  the 
charge  communicated  to  the  protein  particle  by  the  hydrogen  ion  of  the 
acid  is  neutralized  by  the  anion  of  a  precipitating  salt,  what  will  happen 
to  the  cation  of  the  precipitating  salt?  Its  charge  must  be  neutralized, 
and  this  can  only  be  accomplished  by  its  union  with  the  protein  com- 
plex or  else  by  its  union  with  the  acid  radical  which  is  also  attached  to 
the  protein.  In  the  first  alternative,  the  train  of  events  would  be 
represented  by  the  equation: 

2  Protein  HC1      +     Na2SO4      =     (Protein  HCl)2Na2SO4 

and  in  the  second  alternative  by  the  equation: 

2  Protein  HCl     +     Na2SO4      =     Protein2H2SO4     +     2NaCi 

In  other  words,  from  whatever  point  of  view  we  may  regard  the 
precipitation  of  proteins  by  inorganic  salts,  as  soon  as  we  examine 
closely  the  details  of  the  process,  it  becomes  indistinguishable,  by  any 
criterion  which  we  at  present  possess,  from  a  chemical  reaction,  and  it 
seems  to  be  quite  unnecessary  to  invent  a  special  hypothesis  to  account 
for  this  particular  type  of  chemical  reaction,  the  need  of  which  is  not 
experienced  in  interpreting  any  other  of  the  immense  variety  of  chemi- 
cal reactions  yielding  precipitates  with  which  we  are  familiar. 

It  remains,  however,  to  account  for  the  peculiar  relationship  of 
precipitating  power  to  the  valency  of  the  precipitating  ion  which  led 
to  the  elaboration  of  Whetham's  hypothesis.  Now  this  hypothesis, 
when  carefully  examined,  is  seen  to  consist  in  nothing  more  than  a 
restatement,  in  terms  of  the  probabilities  of  molecular  collisions,  of  the 
Guldberg  and  Waage  Mass-law  which  applies  to  all  chemical  reactions. 
According  to  this  law,  the  velocity  with  which  any  given  chemical 
reaction  proceeds,  varies  directly  as  the  active  masses  of  each  of  the 
reacting  molecules.  In  the  case  under  consideration,  presuming  that  a 
given  number  (e.  g.  one)  of  molecules  of  protein  react  with  one  molecule 
of  a  salt  of  a  monovalent  metal  to  form  a  compound,  then  twice  as  many 
molecules  of  the  protein  may  be  supposed  to  react  with  a  molecule  of  a 
salt  of  a  divalent  metal,  and  three  times  as  many  with  a  salt  of  a  triva- 
lent  metal.  Assuming  that  the  active  mass  of  the  colloid  (the  molecu- 
lar concentration  multiplied  by  the  degree  of  electrolytic  dissociation) 


PRECIPITATION  OF  PROTEINS  BY  INORGANIC  SALTS          163 

is  the  same  in  each  of  these  cases  (which  is  also  assumed  in  Whetham's 
theory)  and  equal  to  A,  calling  the  initial  velocities  of  the  respective 
reactions  vi,  v2  and  v3  and  the  concentrations  of  the  mono-  di-  and 
trivalent  ions  Ci,  c2  and  c3  we  have: 

vi  is  proportional  to  A*CI 
V2  is  proportional  to  A2C2 
vs  is  proportional  to  A3cs 

whence  it  follows  that  if  Vi  =  v2  =  v3  and  the  velocity-constants  of 
the  three  reactions  are  equal  (which  is  also  assumed  in  Whetham's 
hypothesis)  : 


and 


1   :      i         i    =    1        A        A2 

Ci        C2         C3 


-  ,         -          and 
Ci  C2 


i.  e.,  the  dilutions  of  the  mono-  di-  and  trivalent  ions  at  which  combina- 
tion proceeds  with  equal  velocity,  are  related  to  one  another  in  the 
same  way.  Now  in  the  experiments  described  above,  -^  is  defined  as  pi 
the  precipitating-power  of  the  salt,  hence: 

PI  i  P2  :  PS     =     1    •    A    :    A2 

which  is  exactly  the  relation  deduced  by  Whetham.  The  experimental 
relations  found  by  Schultz,  Linder  and  Picton,  Hardy  and  others  are, 
therefore,  just  as  explicable  upon  the  assumption  that  the  colloid  reacts 
chemically  with  the  precipitating  ion  as  upon  the  assumption  that  the 
precipitating  ion  acts  in  a  purely  physical  way  through  altering  the 
electrical  condition  of  the  colloidal  particles.  The  former  view  attrib- 
utes to  the  colloids  in  general,  and  to  the  proteins  in  particular  no 
especial  qualities  which  differentiate  them  from  other  chemical  systems, 
while  the  latter  view  necessitates  radical  assumptions  regarding  the 
nature  of  colloidal  solutions  which  have  hitherto  proved  incapable  of 
verification. 

It  will  be  noticed,  however,  that  the  factor  that  determines  the 
precipitating-power  of  a  salt  is  the  velocity  with  which  it  combines, 
with  the  protein  and  not  the  final  equilibrium  which  is  attained.  This 
is  not  surprising  when  we  recollect,  firstly,  the  enormous  part  played 
by  the  velocity  of  change  in  determining  the  final  physical  condition 
of  a  colloid,  and  secondly,  the  method  by  which  the  "  precipitating- 
powers"  of  salts  are  measured.  Linder  and  Picton,  for  example, 
measured  the  precipitating-power  of  salt  solutions  by  titration,  running 
the  solution  of  the  salt  into  the  solution  of  the  colloid  until  precipitation 
just  began  to  be  perceived.  They  expressly  state  that  unless  the  time 
occupied  in  the  titration  be  kept  approximately  the  same,  serious 
deviations  from  the  "valency  rule"  occur:  "As  a  quantity  of  coagu- 
lant insufficient  to  produce  coagulation  immediately,  will  do  so  in  the 


164  COMPOUNDS  OF  THE  PROTEINS 

course  of  time."  Under  these  conditions,  what  is  actually  measured  is 
the  concentration  of  the  precipitating-agent  which  is  requisite  to  bring 
about  a  given  degree  of  change  (visible  precipitation)  within  a  given 
brief  period,  that  is  to  say,  a  velocity,  and  not  an  equilibrium. 

That  protein,  when  it  is  precipitated  by  inorganic  salts,  actually 
enters  into  combination  with  them  and  carries  down  a  portion  of  the 
precipitating  salt,  has  been  shown  in  a  variety  of  instances.  The  most 
exhaustive  investigations  of  this  character  have  been  those  of  Galeotti 
who  has  employed  electrochemical  methods  of  measuring  the  concen- 
tration of  individual  ions  in  the  solutions.  By  these  means  he  has  been 
able  to  show,  for  example,  that  when  Egg-albumin  is  precipitated  by 
silver  nitrate,  Ag+  and  NOj  ions  are  removed  from  the  solution  and 
precipitated  with  the  protein  in  equivalent  proportions;  in  other  words 
the  protein  combines  with  the  whole  molecule  of  silver  nitrate  to  form 
an  insoluble  compound.  It  has  also  been  shown  that  copper  sulphate 
combines  as  a  whole  with  egg-albumin  to  form  .an  insoluble  compound, 
but  in  alkaline  solutions  in  the  presence  of  an  excess  of  the  salt  this 
precipitate  redissolves  and  the  albumin  is  now  found  to  have  combined 
with  an  excess  of  the  copper,  forming  a  soluble  compound,  while  the 
alkali  takes  up  the  excess  of  sulphuric  acid  thus  set  free. 

The  very  important  observation  has  been  made  by  Pauli  that  abso- 
lutely Electrolyte-free  Egg-albumin,  prepared  by  prolonged  dialysis, 
is  not  ionic  (i.  e.}  does  not  drift  in  an  electric  field)  and  that  under  these 
conditions  it  is  not  precipitable  even  by  heavy  metals.  It  is,  however, 
coagulated  by  highly  concentrated  salts.  In  correspondence  with  this 
it  has  been  shown  by  Rohmann  and  Hirschstein  that  the  amount  of 
silver  nitrate  which  will  combine  with  Casein  to  form  an  insoluble 
compound  is  exactly  equivalent  to  the  amount  of  base  (NaOH) 
previously  combined  with  the  casein,  i.  e.,  to  the  number  of  — COHN— 
-linkages  that  have  been  opened  up  and  ionized  by  union  with  an  inor- 
ganic base. 

When  we  now  turn  from  the  phenomenon  of  Precipitation  to  that  of 
Coagulation  we  meet  with  quite  a  different  series  of  relationships. 
Instead  of  the  Coagulative-power  of  salts  being  determined  primarily 
by  valency,  we  find  that  specific  ions  of  varying  valencies  have  high 
coagulative-powers,  while  others  have  low  coagulative-powers,  and 
these  specific  relationships  are  rather  constant  for  a  wide  series  of 
proteins  and  of  other  colloids.  In  coagulation,  also,  both  ions  of  the 
coagulating-salt  participate  in  determining  coagulative-power,  although 
they  act  in  opposite  senses,  the  cations  coagulating  and  the  anions 
inhibiting  coagulation,  or  else  vice  versa.  Thus,  Pauli  found  that  in 
egg-white  (in  which  the  protein  is  electronegative)  the  cations  of  added 
electrolytes  are  the  active  agents  in  inducing  coagulation,  while  the 
anions  inhibit  coagulation.  In  the  following  table  of  Pauli's  the 
cations  are  arranged  in  ascending  order  of  precipitating-power  from 
left  to  right,  while  the  anions  are  arranged  vertically,  the  weakest 


PRECIPITATION  OF  PROTEINS  BY  INORGANIC  SALTS         165 

inhibitor  coming  first  and  the  strongest  last.  A  (+)  indicates  that 
the  salt  which  results  from  the  union  of  the  cation  and  anion  causes 
coagulation  of  Egg-albumin;  while  a  (  — )  indicates , that  it  does  not. 

Cations. 


Anions.  Mg  NH  K  Na  Li 

Fluoride ....  +  .  +  -f- 

Sulphate  .      .  +  +  +  +  + 

Phosphate 

Citrate     . 

Tartrate 

Acetate    . 

Chloride 

Nitrate    . 

Chlorate 

Bromide 

Iodide .... 

Thiocyanate.      ... 

When  the  protein  is  combined  with  acid,  however,  or  is  "electro- 
positive," the  order  of  effectiveness  of  the  different  salts  in  bringing 
about  coagulation  is  exactly  the  reverse  of  the  order  of  effectiveness  in 
bringing  about  the  coagulation  of  protein  combined  with  alkali  ("elec- 
tronegative") protein.  The  series  is  reversed  in  every  respect;  the 
anions  now  induce  coagulation  and  the  cations  inhibit  it.  The  anions 
coagulate  in  the  order: 

CNS  >  I   >  Br  >   NO3  >  Cl  >  CH3COO 

while  the  cations  inhibit  coagulation  in  the  order: 

Li  >  Na  >  K  >  NH4  >   Mg 

We  have  seen  that,  in  order  that  Precipitation  of  a  protein  by  salts 
may  occur,  the  protein  must  be  ionized,  but  for  Coagulation  this  condi- 
tion is  not  requisite.  In  determining  the  rate  of  precipitation  the 
valency  of  the  precipitating  ion  is  of  prime  importance;  in  determining 
the  rate  of  coagulation  it  is  of  comparatively  subordinate  importance. 
For  precipitation  very  low  concentrations  of  the  precipitating  salt 
suffice,  intermediate  concentrations  frequently,  and  indeed  usually 
redissolve  the  precipitate,  and  for  coagulation  high  concentrations  of 
the  salt  are  required.  This  latter  fact,  and  the  fact  that  the  presence 
of  coagulating-salts  aids  coagulation  by  alcohol  and  by  heat,  suggests, 
as  it  did  to  Hofmeister,  that  coagulation  is  dependent  upon  Dehydration 
of  the  protein. 

Starting  from  the  observation  of  Jones  and  Ota,  that  certain  salts 
when  dissolved  in  water,  produce  an  abnormal  depression  of  the. 
freezing-point,  H.  C.  Jones  and  his  pupils  have  built  up  a  very  large 
body  of  evidence  for  the  existence  of  hydrates  (or  "  solvates")  of  sub- 
stances in  solution.  These  investigators  find  that  both  ions  and  undis- 


166  COMPOUNDS,  OF  THE  PROTEINS 

sociated  molecules  can  form  "solvates,"  and  that  these  hydrates  or 
"solvates"  are  readily  decomposed  at  temperatures  which  approach 
the  boiling-point  of  the  solvent,  and  by  the  presence  of  other  agents  in 
the  solution  which  compete  for  the  solvent.  The  determination  of  the 
quantity  of  water  bound  in  this  way  by  substances  in  aqueous  solution, 
is  frequently  a  matter  of  difficulty  and  uncertainty,  but  the  existence 
of  such  "solvate"  compounds  may  be  demonstrated  in  a  variety  of 
ways,  although  their  quantitative  composition  remains,  in  general, 
unknown.  A  very  striking  experiment  which  illustrates  the  formation 
of  "  solvates"  is  that  cited  by  Pickering.  If  a  mixture  of  Propyl  Alcohol 
and  water  be  placed  in  a  semipermeable  vessel  and  surrounded  with 
water,  it  is  found  that  water  enters  the  cell,  but  that  no  propyl  alcohol 
escapes.  If,  however,  the  same  semipermeable  vessel,  containing  the 
same  mixture  of  propyl  alcohol  and  water,  be  immersed  in  propyl 
alcohol,  propyl  alcohol  enters  the  cell  and  water  does  not  leave  it.  In 
other  words,  the  vessel  is  permeable  to  either  propyl  alcohol  or  water 
when  these  are  pure,  but  it  is  impermeable  to  mixtures  of  the  two,  the 
inference  being  that  large  molecular  complexes  are  formed  on  mixing 
these  reagents  which  cannot  pass  through  the  pores  of  the  vessel.  From 
these  and  similar  experiments  Poynting  concludes  that  osmotic  pressure 
is  an  expression  of  the  diminution  in  the  active  mass  of  the  solvent  due 
to  the  formation  of  compounds  with  the  dissolved  substance. 

It  is  a  familiar  fact  to  chemists  that  anhydrous  Cobalt  Chloride 
is  blue,  but  that  on  taking  up  water  it  becomes  violet  or  red.  Ostwald 
believed  that  the  undissociated  cobalt  chloride  molecule  is  blue,  while 
the  cobalt  ion  is  red.  Since,  however,  the  color  of  a  concentrated 
solution  of  cobalt  chloride  can  be  changed  from  purplish-red  to  blue 
by  the  addition  of  relatively  small  amounts  of  calcium  salts,  or  still 
smaller  amounts  of  aluminium  chloride,  or  by  the  addition  of  a  few  drops 
of  alcohol,  it  is  more  probable  that  this  change  in  color  is  due  to  dehy- 
dration of  the  cobalt  chloride  molecule  in  solution,  by  the  abstraction 
of  water  from  it  by  the  added  substance.  Similarly  the  progressive 
change  in  color  of  Cupric  Chloride  solutions,  from  blue  to  greenish-brown, 
on  concentration  or  dehydration  is  attributed  to  the  loss  of  water  on  the 
part  of  cupric  chloride  water-complexes.  G.  N.  Lewis  finds  that  if 
various  bromides  be  added  to  concentrated  solutions  of  Cupric  Bromide 
the  copper  salt  is  dehydrated  (turned  brown)  by  the  salts  of  mono- 
valent  metals  in  the  order:  Li  >  Na  >  NEU  >  K.  Divalent  metals 
dehydrate  more  strongly,  the  order  being:  Mg  >  Ca  >  Sr  >  Ba  while 
trivalent  metals  (Al)  act  still  more  energetically.  The  resemblance 
between  the  order  of  effectiveness  of  the  monovalent  metals  in  dehy- 
drating cupric  bromide  and  their  order  of  effectiveness  in  coagulating 
"electronegative"  protein  is  very  evident. 

The  peculiar  interest  to  the  biological  chemist  of  the  possibility  thus 
indicated,  that  substances  dissolved  in  water  form  loose  combinations 
with  the  solvent,  lies  in  the  especial  significance  of  water  in  relation  to 


PRECIPITATION  OF  PROTEINS  BY  INORGANIC  SALTS       16? 

the  protein  and  polypeptide  structure.     Dehydration  of  a  protein  may 
result  in  one  or  more  of  the  following  series  of  reactions: 


NHs.OOC.R.NHsOH 

+     H2O 

HOHsN  3OOH 

NHs.OOC.R.NHsOH  /NHOC.R.NH3OH 

+    H2O 


NHOC.R.NHaOH  /NHOC.R.NH2 

=     R\  +    H2° 


NHOC.R.NH2  /NHOC.R.NH 

=     R  +    H2° 


and  hydration,  of  course,  may  result  in  the  reversion  of  this  series  of 
changes. 

That  proteins  may  be  thrown  out  of  solution  in  two  very  different 
conditions  of  hydration  is  very  clearly  shown  by  the  following  experi- 
ments : 

Anhydrous  Casein  dissolves  readily  in  cold  anhydrous  Formic  Acid; 
still  more  readily  in  hot  formic  acid.  If,  to  a  two  per  cent,  solution  of 
casein  in  formic  acid,  we  add  a  fairly  concentrated  solution  of  Cupric 
Chloride,  the  mixture  is  at  first  green,  indicating  the  presence  of  lower 
hydrates  of  cupric  chloride,  but  on  adding  more  of  the  solution  it 
becomes  blue,  and  simultaneously  with  the  appearance  of  a  pure  blue 
color,  but  not  before,  precipitation  of  cupric  caseinate  occurs.  If, 
to  five  c.c.  of  a  two  per  cent,  solution  of  casein  in  formic  acid,  we  add 
1J,  2  or  2J  c.c.  of  a  saturated  solution  of  cupric  chloride,  no  precipita- 
tion of  the  caseinate  occurs,  but  on  diluting  this  mixture  with  water  a 
precipitate  results,  and  the  appearance  of  this  precipitate  coincides 
with  the  attainment  of  a  clear  blue  color  on  the  part  of  the  mixture. 

About  six  cubic  centimeters  of  water  are  required  to  produce  a 
permanent  precipitate.  This  precipitate  redissolves  on  heating,  and 
the  mixture  simultaneously  becomes  green;  on  cooling  the  blue  color 
reappears  and  with  it  the  precipitate.  If  formic  acid  be  added  to  the 
mixture  the  precipitate  redissolves  as  soon  as  the  mixture  becomes 
green.  If  the  precipitate  be  very  slight  it  will  redissolve  on  adding 
alcohol.  It  cannot  be  urged  that  the  formation  of  cupric  caseinate 
requires  the  presence  of  more  cupric  ions  than  are  present  in  green 
solutions,  because  green  solutions  of  cupric  chloride  contain  an  abun- 
dance of  ions,  and  casein  will  react  with  very  small  amounts  of  metal 


168  COMPOUNDS  OF  THE  PROTEINS 

ions,  for  although  it  is  itself  insoluble  it  will  drive  carbonic  acid  out  of 
the  sparingly  soluble  calcium  carbonate  to  form  a  freely  soluble  casein- 
ate  of  calcium. 

If  instead  of  adding  water  to  a  mixture  of  five  c.c.  of  two  per  cent, 
casein  in  formic  acid,  and  two  c.c.  of  saturated  cupric  chloride,  we  add 
alcohol;  no  coagulation  occurs  until  the  mixture  changes  in  color  from 
green  to  brown,  wrhen  a  Coagulum  of  cupric  caseinate  is  produced  which 
redissolves  on  adding  water. 

Similar  results  are  obtained  when  a  2-molecular  solution  of  Cobalt 
Chloride  is  employed  instead  of  a  saturated  solution  of  cupric  chloride. 
If  to  five  c.c.  of  a  two  per  cent,  solution  of  casein  in  formic  acid  we  add 
two  to  three  c.c.  of  this  cobalt  chloride  solution,  we  obtain  a  blue- 
purple  mixture.  On  adding  water  to  this  mixture  it  changes  in  color 
from  blue-purple,  through  red-purple  to  clear  pink.  Not  until  a  pure 
pink  color  is  obtained  does  a  precipitate  result.  If,  instead  of  adding 
water,  we  add  a  considerable  volume  of  alcohol  (ten  volumes)  the  mix- 
ture rather  abruptly  changes  to  a  clear  pale  blue,  and  then,  but  not 
before,  we  obtain  a  coagulum  of  cobalt  caseinate. 

Electronegative  casein  (i.  e.,  casein  dissolved  in  alkalies)  is  not 
precipitated  by  the  salts  of  the  alkalies,  although  it  is  readily  precipi- 
tated by  salts  of  the  alkaline  earths.  Electropositive  casein  (i.  e., 
casein  dissolved  in  acids)  is,  however,  very  readily  precipitated  by  salts, 
and  these  precipitates  are  not  soluble  upon  dilution.  Thus  if  two  c.c. 
of  tenth  normal  hydrochloric  acid  be  added  to  five  cubic  centimeters  of 
a  one  per  cent,  solution  of  casein  in  0.008  N.  potassium  hydroxide,  a 
clear,  acid  solution  of  casein  results.  The  casein  is  precipitated  from 
this  solution  by  the  addition  of  four  drops  of  a  saturated  solution  of 
sodium  chloride,  or  by  one  drop  of  a  saturated  solution  of  ammonium 
sulphate.  This  latter  precipitate  does  not  dissolve  on  diluting  the 
mixture  to  one-sixteenth. 

Casein  Formate  affords  no  exception  to  the  rule  that  salts  of  casein 
with  acids  are  precipitable  by  relatively  small  concentrations  of  neutral 
salts,  but  the  precipitation  will  only  occur  'in  the  presence  of  a  sufficiency  of 
water.  If  to  five  cubic  centimeters  of  a  two  per  cent,  solution  of  casein 
in  formic  acid  we  add  a  saturated  solution  of  ammonium  sulphate, 
three  cubic  centimeters  of  this  solution  just  suffice  to  produce  a  coagu- 
lum, this  becomes  more  abundant  on  adding  water,  and  redissolves  on 
adding  formic  acid.  If,  however,  instead  of  adding  three  we  add  two 
cubic  centimeters  of  the  saturated  ammonium  sulphate  solution,  a 
clear  solution  is  obtained.  On  adding  water  to  this  a  precipitate  results 
which  redissolves  on  heating  and  reappears  on  cooling. 
Analogous  results  may  be  obtained  with  Ovomucoid. 
It  is  clear,  therefore,  that  protein  may  be  thrown  out  of  solution 
by  electrolytes  in  two  grades  of  hydration,  the  one  of  high,  the  other  of 
very  low  hydration.  The  former  process  is  what  we  have  termed 
Precipitation,  the  latter  we  have  defined  as  Coagulation.  At  grades  of 


PRECIPITATION  OF  PROTEINS  BY  INORGANIC  SALTS          169 

hydration  intermediate  between  these  extremes  the  protein  may  be 
soluble.  Dehydration,  partial  or  complete,  leading  to  resolution  or  to 
coagulation  may  be  induced  by  heat,  by  non-electrolytes  possessing 
an  affinity  for  water,  or  by  concentrated  electrolytes. 

The  importance  of  a  high  degree  of  dehydration  in  the  production  of 
Coagula,  irresistibly  suggests  that  this  phenomenon  is  dependent  upon 
the  formation  of  anhydrides,  analogous  to  the  Diketopiperazines  which 
may  be  formed  from  the  amino-acids  and  polypeptides  by  dehy- 
drating-agents,  and  of  the  general  formula: 


NH  xlNXl.V^V^V 

R<        |  or        R<^ 

?O.HN' 


Such  bodies  may  exist  either  in  the  keto-form,  illustrated  by  the 
above  formulae,  or  in  the  enol-form,  such  as: 

,N.(HO)Cx 


Coagulation  by  mineral  salts  appears  invariably  to  be  accompanied 
or  preceded  by  chemical  interaction  of  the  coagulating-salt  and  the 
protein  salt  of  an  acid  or  base  which  preexisted  in  solution  before 
the  coagulant  was  added.  The  coagulated  protein  in  these  instances, 
therefore,  does  not  represent  the  unaltered  protein  salt  as  it  existed 
in  solution  before  the  coagulant  was  added.  When  Alcohol  is  used  as 
the  coagulant  however,  it  is  found,  at  least  in  the  case  of  the  Caseinates 
of  the  Alkaline  Earths,  that  the  protein  salt  as  such  is  coagulated, 
carrying  down  with  it  the  amount  of  mineral  base  with  which  it  was 
combined  before  the  coagulant  was  employed,  so  that  after  washing 
out  the  alcohol  with  ether,  and  absorbing  the  ether  by  desiccation  over 
sulphuric  acid,  calcium  caseinate  is  obtained  in  the  form  of  a  dry 
powder  which  is  soluble  in  water,  whereas  free  casein  is  insoluble  in 
water.  If  coagulation  by  alcohol  is  attributable  to  dehydration  of  the 
protein,  the  elements  of  water  must  be  contributed  chiefly  by  the 
interaction  of  free  amino-  and  carboxyl-groups  with  the  formation  of 
ring-anhydrides,  and  that  this  should  be  possible  without  disintegration 
of  the  compounds  with  bases  affords  another  indication  that  free 
carboxyl-groups  are  not  responsible  for  the  union  of  proteins  with  bases. 
The  same  considerations  probably  will  be  found  to  apply  to  the 
coagulation  by  alcohol  of  the  compounds  of  proteins  with  acids,  but  as 
yet  these  compounds  have  not  been  so  thoroughly  investigated  from 
this  standpoint  as  the  compounds  of  proteins  with  bases. 


170  COMPOUNDS  OF  THE  PROTEINS 

COMPOUNDS  OF  PROTEINS  WITH  OTHER  PROTEINS. 

When  the  Protamines,  which,  it  will  be  recollected,  are  strongly  basic 
proteins,  are  added  to  weakly  alkaline  solutions  of  other  proteins, 
precipitates  are  formed  which  consist  of  compounds  of  the  protamine 
and  other  protein  employed.  These  compounds,  once  formed,  are 
tolerably  stable,  and  when  precautions  are  taken  to  prevent  admixture 
with  excess  of  protamine  they  are  found  to  be  of  very  constant  com- 
position. These  compounds  were  investigated  by  Hunter  who  found 
that  wiiile  crystallized  egg-albumin,  casein,  hemi-elastin,  gelatin, 
edestin,  heteroalbumose,  protalbumose,  "alkali  albuminate"  and 
histone  sulphate  yield  a  precipitate  in  alkaline  solutions  upon  the  addi- 
tion of  the  protamine  Clupeine.  Elastin-peptone,  deuteroalbumose  histo- 
peptone  and  several  peptides  fail  to  yield  a  precipitate.  On  digestion 
of  these  precipitates  with  Pepsin  the  protamine  is  set  free,  since  the 
protamines  are  indigestible  by  pepsin,  and  the  remainder  of  the 
compound  is  converted  into  proteoses  and  peptones. 

The  compound  of  Clupeine  with  casein  contains  six  per  cent,  of  the 
protamine  while  the  compound  with  hemoglobin  contains  five  per 
cent  of  protamine.  The  compound  of  Salmine  with  edestin  contains 
about  ten  per  cent,  of  the  protamine. 

When  Globin  and  Casein  are  mixed  in  faintly  acid  solution  a  precipi- 
tate of  globin  caseinate  is  formed  which  is  soluble  in  excess  of  acid 
or  in  dilute  alkalies.  The  precipitate  produced  by  admixture  of  an 
excess  of  globin  with  sodium  caseinate  in  solution  contains  about  34.5 
per  cent,  of  casein.  A  compound  of  globin  with  deuteroalbumose  has 
also  been  prepared  by  C.  L.  A.  Schmidt. 

Thymus-histone  combines  with  Hemoglobin,  according  to  af  Ugglas, 
in  the  proportion  of  one  part  of  thymus-histone  to  two  of  hemoglobin, 
and  with  casein  to  form  a  compound  containing  about  thirty  per  cent, 
of  histone. 

A  particularly  interesting  compound  protein  is  the  Hemoglobin 
Caseinate  which  has  been  prepared  by  af  Ugglas.  To  a  solution  of 
casein  in  alkali  an  excess  of  hydrochloric  acid  is  added  until  the  precipi- 
tate of  free  casein  which  is  at  first  formed  is  redissolved.  The  casein 
hydrochloride  is  precipitated  from  this  solution  by  the  addition  of 
sodium  chloride,  and  the  precipitate  redissolved  and  reprecipitated 
until  the  washings  from  the  precipitate  are  perfectly  neutral.  A 
solution  of  this  substance  added  to  an  excess  of  a  solution  of  hemo- 
globin produces  a  precipitate  containing  33  per  cent,  of  casein  and  about 
66  per  cent,  of  hemoglobin.  The  commonly  accepted  molecular 
weight  of  hemoglobin,  originally  deduced  from  its  content  of  iron, 
and  now  confirmed  by  a  variety  of  measurements,  is  about  16,700.  The 
minimal  molecular  weight  of  casein,  calculated  from  the  minimal 
quantity  of  an  alkali  which  will  just  carry  it  into  solution  (see  p.  154), 
is  8800.  It  seems  evident,  therefore,  that  casein  and  hemoglobin 
combine  with  one  another  in  molecular  proportions.  If  the  same  is 


COMPOUNDS  OF  PROTEINS  WITH  OTHER  PROTEINS        171 

true  of  the  compounds  of  the  various  Protamines  with  such  proteins  as 
casein  and  hemoglobin,  the  low  proportion  of  protamine  which  is 
present  in  these  compounds  would  indicate  that  the  protamines  possess 
molecular  weights  in  the  neighborhood  of  800;  much  lower,  that  is, 
than  the  weights  of  the  majority  of  protein  molecules.  This  corre- 
sponds with  their  less  colloidal  character,  the  compound  Salmine  with 
sulphuric  acid,  for  example,  being  freely  diffusible  through  parchment- 
paper,  and  with  the  relatively  few  amino-acids  they  yield  on  hydrolysis, 
reminding  one  of  the  peptones  rather  than  of  the  more  bulky  and 
complex  native  proteins. 

From  a  variety  of  observations  it  appears  extremely  probable  that 
many  of  the  protein  constituents  which  may  be  isolated  from  the 
various  tissues  and  tissue-fluids  do  not  preexist  there,  but  represent 
fractions  split  off  by  chemical  procedures  from  complex  compounds  of 
proteins  with  proteins  which  are  present  in  the  tissue  or  tissue-fluid. 
Thus  W.  B.  Hardy  has  pointed  out  that  in  untreated  Blood-serum  no 
proteins  exist  which  wander  in  an  electrical  field,  but.  as  soon  as  the 
serum  is  acidified  with  acetic  acid,  a  cloud  appears,  which  is  due 
to  partial  precipitation  of  "Insoluble"  Serum-globulin  (the  globulin- 
fraction  of  serum  which  is  insoluble  in  distilled  water).  On  passing 
a  current  through  this  mixture  the  cloud  moves  over  to  the  anode.  If 
the  serum  be  dialyzed  until  all  of  the  serum-globulin  has  been  precipi- 
tated the  remaining  protein  is  now  found  to  be  completely  ionic  and  is 
precipitated,  on  passing  a  current,  at  the  anode.  Dialysis,  therefore, 
or  acidification  of  blood-serum  evidently  accomplishes  the  detachment 
of  a  fraction  ("insoluble"  serum-globulin)  from  the  protein-complex 
which  preexists  in  untreated  serum.  This  fraction  is  electrically 
dissociated  and  so  is  the  remainder  from  which  it  is  split  off,  but  the 
original  protein-complex  is  not  dissociated  at  all. 

Moreover,  as  Hardy  has  also  pointed  out,  the  globulin  which  we 
separate  by  dialysis  or  by  acidification  and  dilution  from  blood  serum 
possesses  very  different  physical  characteristics  from  any  which  are 
displayed  by  the  proteins  in  unmodified  blood-serum.  In  Hardy's 
words:  "The  globulin-fraction  has  an  abiding  characteristic.  In  all 
its  solutions  its  molecular  state  is  so  gross  as  to  cause  the  molecules  to  be 
arrested  by  a  porous  pot.  They  will  not  pass  such  a  filter  even  under 
pressure.  In  this  it  is  sharply  distinct  from  the  parent  serum-protein, 
which  is  readily  filtrable.  If  globulin  be  present  as  such  in  serum  it  is 
not  only  non-ionic,  but  the  agent  which  dissolves  it  must  be  something 
more  than  alkali  and  salt,  since  either  alone  or  together  they  will  not 
produce  so  high  a  grade  of  solution." 

"The  difference  in  the  molecular  grade  of  globulin  when  once 
separated,  and  the  electrical  homogeneity  of  serum-protein  and  of  the 
fraction  (still  capable  of  further  subdividion  by  salting-out)  which 
remains  after  the  alkaline  globulin  fraction  which  most  readily  appears, 
has  been  removed,  suggests  that  serum-protein  is  a  complex  unit.  If 
such  a  unit  exists  it  is  not  saturated  with  globulin.  Fresh  ox-serum  has 


172  COMPOUNDS  OF  THE  PROTEINS 

an  extraordinary  power  of  dissolving  globulin,  it  will  take  up  almost 
its  own  volume  of  the  thick  cake  at  the  bottom  of  a  centrifuge  tube; 
and  in  ox-serum  so  saturated  there  is  not  a  trace  of  alkali-globulin  nor 
of  any  ionic  protein." 

The  phenomena  observed  by  Hardy  appear  to  admit  of  interpretation 
by  the  view  that  the  protein-complex  in  serum  is  formed  by  the  union 
of  a  number  of  alkali-protein  compounds,  the  union  taking  place  in  a 
manner  analogous  to  that  which  occurs  between  proteins  and  inorganic 
salts,  protein-acid  or  protein-base  compounds  in  this  instance  taking 
the  place  of  the  inorganic  salts.  The  soluble  compounds  which  are 
thus  formed  are  non-ionic,  as  evidenced  by  lack  of  motion  in  an 
electrical  field. 

It  has  been  suggested  that  the  tissues  and  tissue-fluids  of  the  various 
species  of  the  animal  kingdom  may  owe  their  specific  and  individual 
character  to  a  characteristic  structure  of  the  protein-complexes  in  their 
tissues  or  tissue-fluids.  We  shall  have  occasion  to  revert  to  this  possi- 
bility in  a  subsequent  chapter  (Chapter  XIV) . 


REFERENCES. 
GENERAL: 

Robertson:     The  Physical  Chemistry  of  the  Proteins.     New  York,  1918. 
MODE  OP  UNION  OF  AMINO-ACIDS  AND  FREE  AMINO-GROUPS  IN  PROTEINS: 

Hofmeister:     Ergeb.  d.  Physiol.  1  Abt.,  1902,  1,  p.  759. 

Levites:     Zeit.  f.  physiol.  Chem.,  1904-5,  43,  p.  202.     Biochem.  Zeit.,  1909,  20,  p. 
224. 

Van  Slyke  and  Birchard:     Jour.  Biol.  Chem.,  1913-14,  16,  p.  539\ 
MODE  or  UNION  OF  ACIDS  AND  BASES  WITH  PROTEINS: 

Bugarszky  and  Liebermann:     Arch.  f.  d.  ges.  Physiol.,  1898,  72,  p.  51. 

Hardy:     Jour,  of  Physiol.,  1905,  33,  p.  251. 

Robertson:     Jour,  of  Physical  Chem.,  1911,  15,  p.  521. 

Blasel  and  Matula:     Biochem.  Zeit.,  1913-1914,  58,  p.  417. 

Pauli  and  Hirschfeld:     Ibid.,  1914,  62,  p.  245. 

Osborne  and  Leavenworth:     Jour.  Biol.  Chem.,  1913,  14,  p.  481. 
PRECIPITATION  AND  COAGULATION: 

Whetham:     Theory  of  Solution.     Cambridge,  1902,  pp.  396  and  398. 

Pauli  and  Handovsky:     Biochem.  Zeit.,  1909,  18,  p.  340;  1910,  24,  p.  239. 

Robertson:     Jour.  Biol.  Chem.,  1911,  9,  p.  303. 
COMPOUNDS  OF  PROTEINS  WITH  OTHER  PROTEINS: 

Hardy:     Jour.  Physiol.,  1905-6,  33,  p.  251,  appendix  p.  327. 

Hunter:     Zeit.  physiol.  Chem.,  1907,  53,  p.  526. 

af  Ugglas:    Biochem.  Zeit.,  1914,  61,  p.  469. 

Schmidt:     Jour.  Biol.  Chem.,  1916,  25,  p.  63.     Univ.  of  California  Pub.  Pathology, 
1916,  2,  p.  157. 


CHAPTER  IX. 
THE  NUCLEIC  ACIDS  AND  THE  NITROGENOUS  BASES. 

THE  DECOMPOSITION-PRODUCTS  OF  THE  NUCLEIC  ACIDS. 

The  nucleic  acids  form  the  prosthetic  group  in  an  important  series 
of  conjugated  proteins,  the  Nucleoproteins.  These  substances  usually, 
but  not  invariably,  occur  in  nuclear  tissues  and  may  be  precipitated 
from  tissue-extracts  by  acidification  with  acetic  acid,,  in  excess  of 
which  they  do  not  dissolve.  The  nucleoproteins  dissolve,  however, 
in  dilute  mineral  acids  and  in  dilute  alkalies;  they  are  not  soluble  in 
distilled  water.  Certain  nucleoproteins  designated  the  /3-nucleopro- 
teins,  however,  are  soluble  in  boiling  water  and  are  extracted  from 
tissues  in  this  manner,  leaving  the  other  tissue-proteins  in  the  form  of 
coagula  in  the  insoluble  residue;  the  jS-nucleoproteirs  are  also  pre- 
cipitable  by  acetic  acid. 

When  the  alkali-compound  of  a  nucleoprotein,  dissolved  in  water, 
is  heated,  a  portion  of  the  protein  is  split  off  in  a  coagulated  form, 
while  the  residue  of  the  molecule,  which  still  contains  protein  but  is 
much  richer  in  phosphorus  than  the  original  nucleoprotein,  remains 
in  solution.  A  similar  cleavage  is  brought  about  by  the  Pepsin  in 
gastric  juice,  which  digests  the  protein  fraction  which  is  split  off  from 
the  nucleoprotein,  but  leaves  a  residue  undigested  which  still  contains 
protein  united  to  nucleic  acid.  This  residue  is  designated  Nuclein. 

By  means  of  more  intense  hydrolysis  with  alkali  the  nucleins  are 
split  up  into  products  of  protein  hydrolysis  and  the  alkali  salts  of 
the  nucleic  acids.  These  salts  may  be  precipitated  from  concentrated 
solutions  by  the  addition  of  alcohol. 

Upon  hydrolysis  with  acids  all  of  the  nucleic  acids  yield  three  widely 
differing  groups  of  products.  In  the  first  place  phosphoric  acid  is  an 
essential  constituent  of  the  molecule,  secondly  a  carbohydrate  radical, 
which  may  be  either  a  pentose  or  a  hexose,  and  thirdly  a  nitrogenous 
base  belonging  to  the  group  of  Purine  Bases  or  to  the  closely  allied 
group  of  Pyrimidine  Bases. 

The  carbohydrate  radical  differs  essentially  in  nucleic  acids  of  different 
origin.  In  all  of  the  plant-nucleic  acids  which  have  been  investigated, 
the  carbohydrate  radical  has  been  found  to  be  a  pentose  d-ribose: 

CHO 
HCOH 

HCOH 
HCOH 
CH;OH 


174          NUCLEIC  ACIDS  AND  THE  NITROGENOUS  BASES 

which,  until  its  discovery  among  the  decomposition-products  of  nucleic 
acids,  was  unknown  in  nature.  It  is  now  not  only  recognized  as  the 
carbohydrate  radical  of  plant  nucleic  acid,  but  also  regarded  as  the  only 
pentose  which  normally  occurs  in  animal  tissues.  In  two  nucleic 
acids  found  in  animal  tissues,  but  possibly  traceable  to  a  vegetable 
origin,  namely  Inosinic  Acid  and  Guanylic  Acid,  d-ribose  also  constitutes 
the  carbohydrate  radical,  but  the  nucleic  acid  which  is  most  charac- 
teristic of  animal  tissues,  Thymus  Nucleic  Acid,  so  called  because  of 
the  circumstance  that  it  was  first  prepared  in  a  pure  condition  from 
the  tissues  of  the  thymus,  yields  Levulinic  Acid  on  hydrolysis  by  acids. 
Now  levulinic  acid,  or  /3-acetyl  propionic  acid: 

CH3CO.CH2.CH2.COOH 

is  formed  when  Hexoses  are  boiled  with  mineral  acids,  Formic  Acid 
being  produced  at  the  same  time: 

C6Hi2O6     =     C6H8O3     +     HCOOH     +     H2O 

In  the  hydrolysis  of  thymus  nucleic  acid  by  mineral  acids,  formic 
acid  is  produced  as  well  as  levulinic  acid.  It  is  evident,  therefore, 
that  both  of  these  products  are  derived  from  a  Hexose  radical  in  the 
nucleic  acid  and  confirmation  of  this  inference  is  supplied  by  the  fact 
that  on  oxidation  of  thymus  nucleic  acid  with  nitric  acid,  Saccharic 
Acid  is  included  among  the  products,  and  saccharic  acid  must  have  a 
hexose  precursor: 

2C6Hi2O6-}-3O2  =2C6HioO8+2H2O 

Among  the  Nitrogenous  Bases  which  result  from  the  acid  hydrolysis 
of  nucleic  acids,  Guanine  and  Adenine,  which  are  Purine  Bases,  are 
found  in  both  animal  and  plant  nucleic  acids,  but  among  the  Pyrimidine 
Bases  which  are  yielded  by  the  two  classes  of  nucleic  acid,  there  is  a 
difference,  for  while  both  types  of  nucleic  acid  yield  Cytosine,  the  animal 
nucleic  acid  ("thymus  nucleic  acid")  yields  Thymine,  and  vegetable 
nucleic  acid  yields  Uracil. 

The  pyrimidine  bases  are  heterocyclic  compounds  which  are  dis- 
tinguished by  the  possession  of  the  following  nucleus: 

I 


Pyrimidine  itself  has  the  formula: 

N 


HC 


DECOMPOSITION-PRODUCTS  OF  THE  NUCLEIC  ACIDS       175 

It  does  not  occur  among  the  decomposition-products  of  the  nucleic 
acids.  Its  derivatives  Uracil,  Cytosine  and  Thymine  have  the  following 
formulae : 

HN CO  N=C.NH2  HN CO 

|  I 

OC         CH  OC         CH  OC         C.CH3 

I          II 


HN CH  HN CH  HN CH 

Uracil.  Cystosine.  Thymine. 


Uracil  is  therefore  dioxypyrimidine,  cytosine  is  amino-oxypyrimidine 
and  thymine  is  methyluracil.  Cytosine  is  transformed  into  uracil  by 
the  action  of  nitrous  acid. 

Each  of  these  bases  has  been  prepared  synthetically;  they  are 
known  to  occur  in  Nature,  however,  only  as  decomposition-products 
derived  from  nucleic  acids  by  hydrolysis.  They  are  sparingly  soluble 
in  cold  water,  more  soluble  in  hot  water.  Cytosine  dissolves  in  alcohol, 
uracil  with  difficulty,  and  thymine  not  at  all.  Cytosine  and  thymine 
are  precipitated  by  phosphotungstic  acid.  Uracil  is  not.  On  heating, 
thymine  sublimes  without  decomposition,  uracil  partly  decomposes  and 
partly  sublimes,  while  cytosine  undergoes  decomposition. 

Cytosine  and  uracil  give  the  Weidel  Reaction  as  follows:  To  a 
small  quantity  of  solution  chlorine  water  is  added,  and  the  mixture 
boiled.  The  solution  is  evaporated  to  dryness  and  then  exposed  while 
warm  to  the  vapors  of  ammonia.  A  purple-red  color  develops.  This 
reaction  is  frequently  referred  to  as  the  Murexide  Reaction  because  it 
is  due  to  the  formation  of  Ammonium  Purpurate  which  is  believed  to 
be  identical  with  the  scarlet  dye  found  in  the  mollusc  murex  which 
furnished  the  "purple"  of  the  ancient  Romans.  An  intermediate  stage 
in  the  reaction  is  the  formation  of  Alloxan. : 

HN CO 

oc       co 

HN CO 

Alloxan. 

and  the  test  is  only  given  by  such  substances  as  can  be  made  to  yield 
alloxan  by  oxidation.  The  reaction  is  therefore  not  infrequently 
alluded  to  as  the  Alloxan  Reaction.  Nitric  acid  may  be  used  in  the 
place  of  chlorine  as  the  oxidizing  agent. 

Cytosine  and  uracil  also  give  Wheeler  and  Johnson's  Reaction:  To 
the  solution  of  the  substance  bromine  water  is  added  drop  by  drop 
until  a  permanent  cloudiness  appears.  Baryta  water  is  then  added, 


176  NUCLEIC  ACIDS  AND  THE  NITROGENOUS  BASES 

when  a  purple  or  violet  precipitate  appears.    The  Purine  Bases  are 
formed  by  the  union  of  a  pyrimidine  nucleus  with  an  Iminazolyl  radical : 

N=CH  N=CH 

HC NHx 

HC        CH 


N CH 

Purine.  Pyrimidine. 

The  purine  bases  which  are  obtained  from  the  nucleic  acids  repre- 
sent only  two  members  of  a  large  group  of  substances  which  includes 
Uric  Acid,  Caffeine  and  Theobromine.  For  convenience  of  reference 
the  carbon  and  nitrogen  atoms  in  the  central  complex  are  often  num- 
bered as  follows: 


> 

r/ 


N3 


The  purine  substances  which  are  most  important  from  a  physio- 
logical point  of  view  are  Uric  Acid,  Xanthine,  Guanine,  Hypoxanthine 
and  Adenine,  while  Caffeine,  Theobromine  and  Theophylline  are  also  of 
importance  from  a  medical  and  dietetic  point  of  view.  Their  structure 
is  as  follows: 

Uric  acid  is  2,  6,  8,  trioxypurine 

Xanthine  "  2,  6,  dioxypurine 

Guanine  "  2,  amino,  6,  oxypurine 

Hypoxanthine  "  6,  oxypurine 

Adenine  "  6,  aminopurine 

Caffeine  "  1,  3,  7,  trimethyl,  2,  6,  dioxypurine 

Theobromine  "  3,  7,  dimethyl,  2,  6,  dioxypurine 

Theophylline  "  1,  3,  dimethyl,  2,  6,  dioxypurine 

Thus  the  formula  for  Guanine  may  be  graphically  represented: 
HN co 


H2NC 


N 

Guanine 

while  that  of  Adenine  is  as  follows: 

N=CNH2 


HC 


DECOMPOSITION-PRODUCTS  OF  THE  NUCLEIC  ACIDS        177 

The  purine  bases  are  all  precipitable  from  acid  solutions  by  phos- 
photungstic  acid  or  from  ammoniacal  solutions  by  silver  nitrate. 
Guanine  is  insoluble  in  water,  alcohol  or  ether,  but  readily  dissolves  in 
dilute  acids  or  alkalies  (with  the  exception  of  ammonia).  It  does 
not  give  Weidel's  reaction,  but  with  nitric  acid  it  yields,  on  evaporation, 
a  yellow  residue  wThich  turns  bluish- violet  on  heating  with  sodium 
hydroxide.  With  chlorine  water  guanine  decomposes,  yielding  Guani- 
dine,  Parabanic  Acid  and  carbon  dioxide: 

HN CO 

I         I 

H2NC         C NH\  /NH2      HN— CO 

/  I 


+  3O   +  H  2O    =   HN=C<  +   OC 

\  I        I 


C02 


XNH2      HN— CO 

Guanine.  Guanidine.        Parabanic  acid. 


Free  guanine  is  found  in  the  scales  and  swimming-bladder  of  fishes. 
It  also  occurs  occasionally  in  the  form  of  concretions  in  the  retinal 
epithelium  of  fishes  and  in  the  joints  of  pigs  suffering  from  "guanine 
gout."  It  forms  an  important  constituent  in  the  excrement  of 
spiders. 

Guanine  is  hydrolyzed  by  an  enzyme,  Guanase  which  is  found  in  a 
variety  of  tissues,  particularly  those  of  the  pancreas  and  thymus  (but 
not  the  Spleen).  The  products  are  Xanthine  and  ammonia: 

HN CO 

H2NC         C NH\ 

II      II         \ 

>CH     +     H2O      = 


HN 

Guanine.  Xanthine. 


Adenine  undergoes  an  analogous  change  with  the  production  of 
Hypoxanthine,  but  the  enzyme  which  brings  this  about  (Adenase) 
appears  to  be  a  different  one  from  that  which  accomplishes  the  deamini- 
zation  of  guanine,  since  it  occurs  in  the  spleen,  from  which  guanase  is 
absent.  Hence  when  the  tissues  of  the  thymus  or  pancreas  are  allowed 
to  undergo  Autolysis;  that  is  to  say,  spontaneous  hydrolysis  by  their 
own  enzymes,  the  purines  which  are  isolated  from  the  mixture  are 
the  oxypurines,  xanthine  and  hypoxanthine,  instead  of  the  amino- 
purines,  guanine  and  adenine  which  result  from  hydrolysis  by  acids. 

Adenine  is  sparingly  soluble  in  cold,  but  readily  soluble  in  hot  water; 
it  is  readily  soluble  in  acids  and  alkalies.  Adenine  does  not  give 
Weidel's  reaction.  With  nitric  acid,  on  evaporation,  it  gives  a  nearly 
12 


178          NUCLEIC  ACIDS  AND  THE  NITROGENOUS  BASES 

colorless  residue  which  does  not  turn  red  or  violet  on  heating  with 
alkali.  With  hydrochloric  acid  and  zinc  and  subsequent  addition  of 
alkali  an  adenine  solution  yields  a  ruby-red  color  which  changes  to  a 
brownish  tinge.  Adenine  has  been  obtained  from  certain  pathological 
urines  (leukemia)  and  it  occurs  in  considerable  amounts  in  tea-leaves. 

THE  STRUCTURE  OF  THE  NUCLEIC  ACIDS. 

The  nucleic  acid  of  yeast  appears  to  be  identical  with  the  nucleic 
acid  of  the  wheat-kernel,  Tritico-nucleic  Acid.  It  yields,  on  complete 
hydrolysis,  two  purine  bases  and  two  pyrimidine  bases,  namely, 
guanine,  adenine,  cytosine  and  uracil. 

When  yeast  nucleic  acid  is  heated  in  neutral  solutions  under  pressure 
to  175°  C.  it  splits  off  phosphoric  acid  and  yields  four  different  Nucleo- 
sides  each  consisting  of  a  molecule  of  purine  or  pyrimidine  base  united 
to  a  molecule  of  a-ribose.  These  nucleosides  are  the  following: 


Guanosine       .....      ......... 

Adenosine       .............. 

Cytidine    ............... 

Undine      ...........     ....     CBH9O4.C4H3N2O2 

It  follows  that  in  the  undecomposed  molecule  of  nucleic  acid  the 
purine  and  pyrimidine  bases  must  be  attached  directly  to  the  a-ribose 
molecules.  The  nucleosides  do  not  reduce  Fehling's  Solution  and 
hence  the  carbohydrate  radical  must  be  united  to  the  basic  radical  in 
such  a  way  as  to  involve  destruction  of  the  actual  or  potential  aldehyde 
structure  of  the  sugar,  in  other  words  these  compounds  are  analogous 
to  the  Glucosides. 

If,  instead  of  hydrolyzing  nucleic  acid  with  the  aid  of  heat  or  inor- 
ganic catalyzers,  we  employ  extracts  of  various  organs,  such  as  the 
kidney,  heart-muscle,  liver,  pancreas,  or  intestinal  mucosa,  or  if  we 
employ  blood-serum  or  hemolyzed  blood,  all  of  which  contain  the 
enzyme  Nuclease,  the  nucleic  acid  is  split  into  four  different  Mono- 
nucleotides  each  of  which,  on  intense  hydrolysis,  yields  phosphoric  acid, 
a  carbohydrate  which  in  the  case  of  yeast  nucleic  acid  is  a-ribose,  and 
one  of  the  four  different  purine  and  pyrimidine  bases  which  the  original 
molecule  contained.  The  molecule  of  nucleic  acid  is,  therefore,  a 
Tetranucleotid,  built  up  out  of  the  union  of  four  mononucleotid 
radicals. 

Two  mononucleotids  are  known  to  occur  in  animal  tissues,  they  are 
Guanylic  Acid,  obtained  by  the  partial  hydrolysis  of  /3-nucleoproteins, 
those  nucleoproteins  which  may  be  extracted  from  a  variety  of  tissues 
by  Boiling  Water,  and  Inosinic  Acid  which  exists  as  such  in  most 
extracts. 

When  guanylic  acid  is  completely  hydrolysed  by  mineral  acids  it 
yields  phosphoric  acid,  a-ribose  and  guanine.  It  yields  no  other 
purine  base  and  no  pyrimidine  bases.  By  means  of  hydrolysis  in 


STRUCTURE  OF  THE  NUCLEIC  ACIDS  179 

neutral  water  under  pressure,  phosphoric  acid  may  be  split  off  from 
this  substance  and  Guanosine  or  the  nucleoside  of  guanine  is  produced. 
The  /3-Nucleoproteins  are,  therefore,  compounds  of  protein  with  a 
mononucleotid,  while  the  normal  or  a-Nucleoproteins  are  compounds 
of  protein  with  a  tetranucleotid. 

Inosinic  Acid  is  prepared  from  meat-extracts  by  converting  it  into 
the  barium  salt  which  is  very  sparingly  soluble  in  water.  On  hydrol- 
ysis with  acids  it  yields  phosphoric  acid,  a-ribose  and  Hypoxanthine 
in  molecularly  equivalent  proportions.  It  will  be  recollected  that 
hypoxanthine  may  be  derived  from  adenine  by  simple  deaminization : 


N C N  '  )CE      +     NH3 

N C r* 

Adenine.  Hypoxanthine. 

so  that  inosinic  acid  is  a  derivative  of  a  simple  mononucleotid  con- 
taining adenine.  The  fact  that  the  mononucleotids  in  animal  tissues 
yield  a-ribose  on  hydrolysis  while  the  tetranucleotid,  Thymus  Nucleic 
Acid,  which  is  characteristic  of  animal  tissues  yields  levulinic  acid 
winch  must  be  derived  from  a  hexose  radical,  leads  us  to  infer  that 
the  mononucleotids  which  are  found  in  animal  tissues  are  derived 
from  a  vegetable  source  and  are  possibly  not  synthesised  by  animal 
tissues  at  all,  but  formed  by  partial  hydrolysis  and  subsequent  modi- 
fication of  plant  nucleic  acids  received  in  the  food. 

By  very  careful  hydrolysis  with  acids,  interrupting  the  process 
before  it  is  complete,  it  is  possible  to  split  off  hypoxanthine  from 
inosinic  acid,  leaving  a  compound  of  phosphoric  acid  and  pentose.  On 
the  other  hand,  by  neutral  hydrolysis  under  pressure,  phosphoric  acid 
is  split  off  leaving  the  pentose  combined  with  hypoxanthine.  It  is 
evident,  therefore,  that  in  this  mononucleotid  the  carbohydrate  radical 
occupies  a  middle  position,  linking  together  the  phosphoric  acid  on 
the  one  hand  and  the  purine  base  on  the  other.  This  will  be  clear  from 
the  following  schema: 

by  neutral  hydrolysis 
Phosphoric  aci  d — pentose — hypoxanthine 
by  acid  hydrolysis 

we  shall  see  that  the  arrangement  of  the  radical  in  other  mononucleotids 
is  probably  of  the  same  type. 


180  NUCLEIC  ACIDS  AND  THE  NITROGENOUS  BASES 

There  are  three  conceivable  arrangements  of  the  three  constituent 
radicals  of  Guanylic  Acid.  They  are: 

O  00 

||  II  II 

HO— P— O.CsHsOs.CsI^NaO,    C5H9O4.0— P— C5H4N5O,    C5H9O4.C5H3N5O— P— OH 

T  k  9 

Of  these  three  arrangements  II  cannot  be  the  one  which  actually  occurs 
in  guanylic  acid,  because  on  neutral  hydrolysis  under  pressure  it  yields 
Guanosine  C5H9O4.C5H4N5O,  which  would  be  impossible  if,  in  the 
original  molecule,  the  carbohydrate  and  basic  radicals  were  separated 
by  the  interposition  of  phosphoric  acid.  Either  I  or  III  must  be  the 
correct  formula. 

Now  on  comparing  the  rates  at  which  phosphoric  acid  and  guanine 
are  liberated  from  guanylic  acid  by  acid  hydrolysis,  it  is  found  that 
guanine  is  liberated  much  more  rapidly  than  phosphoric  acid.  This 
implies,  of  course,  that  during  the  progress  of  hydrolysis,  while  guanine 
is  being  split  off,  phosphoric  acid  is  being  held  in  combination  with 
some  other  substance  from  which  compound  it  is  detached  with  relative 
difficulty.  The  only  substance,  guanine  being  excluded,  with  which 
the  phosphoric  acid  can  be  combined  is  a-ribose.  It  follows,  there- 
fore, that  phosphoric  acid  is  attached  to  the  molecule  through  the 
pentose  radical,  and  formula  I  must  represent  the  actual  arrangement 
of  the  groups  in  guanylic  acid.  The  same  reasoning  applies  to  the 
adenine-uracil  dinucleotid  which  may  be  split  off  from  yeast  nucleic 
acid  by  partial  enzymatic  hydrolysis.  We  infer,  therefore,  from  these 
facts  and  from  the  general  similarity  of  the  various  mononucleotids 
to  one  another  that  the  arrangement  of  radicals  in  all  of  them  is: 
Phosphoric  acid  —  carbohydrate  —  purine  or  pyrimidine. 

It  remains  to  be  considered  how  these  mononucleotid  radicals  are 
united  together  to  form  the  tetranucleotids  characteristic,  respectively, 
of  vegetable  and  animal  tissues. 

Three  alternative  possibilities  exist,  namely,  (a)  that  the  mono- 
nucleotids are  united  to  one  another  through  their  phosphoric  acid 
groups,  so  that  the  tetranucleotid  would  be  a  substituted  polyphos- 
phoric  acid.  This  was  the  view  originally  propounded  by  Kossel  and 
has  claimed  very  general  acceptance  until  quite  recently;  (6)  that  the 
mononucleotids  are  united  to  one  another  through  their  carbohydrate 
radicals  and  (c)  that  they  are  united  to  one  another  through  their 
purine  or  pyrimidine  radicals.  Between  the  two  latter  alternatives 
it  has  not  as  yet  proved  possible  to  decide  with  certainty,  but  the 
first  alternative,  that  the  mononucleotids  are  united  to  one  another 
through  their  phosphoric  acid  radicals,  may  be  dismissed  for  the 
following  reasons: 


STRUCTURE  OF  THE  NUCLEIC  ACIDS  181 

Yeast  nucleic  acid  is  known  to  consist  of  the  following  four  mono- 
nucleotids. 

H(X 
O    =  P—  O.C6H8O3.C6H4N5 

/ 

HO/ 

Adenine  mononucleotid 

HCk 
O    =  P—  O.C5H8O3.C4H3N2O2 

HO/ 

Uracil  mononucleotid 

HOv 


O    = 

HO/ 

Cytosine  mononucleotid 

HOv 
O    =  P—  O.C5H8O3.CBH4N5O 

HO/ 

Guanine  mononucleotid 

We  know,  also,  that  these  mononucleotids  are  united  to  one  another 
in  the  order  indicated,  for  when  yeast  nucleic  acid  is  heated  with 
ammonia  it  yields  adenine-uracil  dinucleotid,  so  that  the  constituent 
mononucleotids  of  this  substance  must  be  united  together  in  the 
unaltered  nucleic  acid  molecule.  On  the  other  hand,  when  carefully 
heated  with  acids,  yeast  nucleic  acid  splits  off  adenine  and  guanine 
mononucleotids  leaving  uracil-cytosine  dinucleotid.  It  is  evident, 
therefore,  that  the  uracil  and  cytosine  mononucleotid  radicals  are 
united  to  one  another  in  the  yeast  nucleic  acid  molecule,  and  that  the 
adenine  and  guanine  mononucleotids  form  the  extremities  of  the 
molecule. 

Now  the  Adenine  Uracil  Dinucleotid  might  conceivably  consist  of 
two  mononucleotids  united  by  their  phosphoric  acid  radicals,  or  they 
might  be  united  in  some  other  manner.  If  they  were  united  by  their 
phosphoric  acid  molecules,  at  least  one  of  the  hydroxyl-groups  of  the 
phosphoric  acid  radicles  would  disappear  by  neutralization.  The 
total  number  of  available  hydroxyl-groups  contained  in  the  two 
phosphoric  acid  radicals  is  four,  so  that  the  maximum  number  of 
molecules  of  any  base  that  adenin-uracil  dinucleotid  could  combine 
with  would  be  four.  If  any  hydroxyl-groups  were  neutralized  by 
union  of  phosphoric  acid  radicals  with  each  other  or  with  other  parts 
of  the  associated  mononucleotid  the  free  hydroxyl-groups  would  be  less 
than  four,  and  the  dinucleotid  would,  in  consequence,  neutralize  less 
than  four  molecules  of  a  base.  Now  adenine-uracil  dinucleotid  forms 
a  compound  with  four  molecules  of  Brucine.  It  follows,  therefore,  that 


182          NUCLEIC  ACIDS  AND  THE  NITROGENOUS  BASES 

the  phosphoric  acid  radicals  of  adenine  and  uracil  mononucleotids 
are  not  utilized  in  binding  these  constituents  of  the  nucleic  acid  mole- 
cule together. 

When  a  Purine  Nucleotid  is  heated  with  dilute  sulphuric  acid,  phos- 
phoric acid  is  liberated  rapidly  and  completely.  On  the  contrary, 
when  a  Pyrimidine  Nucleotid  is  similarly  treated,  phosphoric  acid  is 
split  off  slowly.  Yeast  nucleic  acid  yields  one-half  of  its  phosphoric 
acid  rapidly,  and  the  remaining  half  slowly.  Now  if  we  compare  the 
relative  rates  of  splitting  off  phosphoric  acid  by  adenine-uracil  dinu- 
cleotid  and  by  the  whole  yeast  nucleic  acid  when  treated  in  this 
manner,  we  find  the  relative  rates  of  yielding  phosphoric  acid  are 
identical.  Hence,  so  far  as  phosphoric  acid  is  concerned,  the  nucleic 
acid  molecule  consists  of  two  symmetrical  parts.  Union  of  the  two 
dinucleotid  fractions  to  form  whole  nucleic  acid  does  not  in  the  slight- 
est degree  affect  the  rate  of  yield  of  phosphoric  acid  by  the  component 
dinucleotids,  and  hence,  phosphoric  acid  cannot  be  concerned  in  their 
union,  and  the  phosphoric-acid  linkage  (2)  in  the  subjoined  diagram 
evidentlv  does  not  exist  in  nucleic  acid. 


(i) 


(2) 


(3) 


The  molecule  of  yeast  nucleic  acid  having  thus  been  shown  to 
consist  of  two  symmetrically  constructed  halves,  so  far  as  phosphoric 
acid  is  concerned,  it  follows  that  if  linkage  (3)  exists,  then  linkage 
(1),  which  would  unite  the  adenine,  and  uracil  mononucleotids  must 
also  exist,  but  this  linkage  has  been  shown  not  to  exist,  by  the  compo- 
sition of  the  brucine  salt  of  the  adenine-uracil  dinucleotid.  Hence 
linkage  (3)  does  not  exist  either  and,  in  short,  no  phosphoric-acid  link- 
ages exist  which  bind  molecules  of  mononucleotid  together  to  form  the 
tetranucleotid  yeast  nucleic  acid. 

Between  the  two  remaining  forms  of  linkage,  by  the  carbohydrate 
or  by  the  purine  or  pyrimidine  radical  it  has  not  yet  been  possible  to 
certainly  decide.  P.  A.  Levene,  however,  concludes  that  in  Cystosine- 
uracil  Dinucleotid  only  two  possibilities  exist,  either  constituent  mono- 
nucleotids are  connected  by  ribose  to  ribose,  or  else  by  uracil  (not  by 
cystosine)  to  ribose.  W.  Jones  believes  yeast  nucleic  acid  to  be 
constituted  as  follows: 


STRUCTURE  OF  THE  NUCLEIC  ACIDS 


183 


O   =  P—  O.C5H7O2.C6H4N6 


0 


O    =  P— O.C5H6O.C4H3N2O2 
HO/ 


O 


II 


O    =  P— O.C5H6O.C4H4N3O 
HO/ 


0 


HOv 

\ 
O    =  P— O.C5H7O2.C6H4N6O 


Yeast  nucleic  acid. 

Tritico-nucleic  Acid  from  the  wheat  embryo  is  identical  in  physical 
behavior  and  in  the  products  it  yields  on  hydrolysis,  with  yeast  nucleic 
acid.  They  are  therefore  believed  to  be  identical  substances,  and  it 
is  considered  probable  that  this  is  the  only  vegetable  tetranucleotid. 

Thymus  Nucleic  Acid,  it  will  be  recollected,  is  yielded  by  the  partial 
hydrolysis  of  all  nucleoproteins  of  animal  origin.  It  contains  a  hexose 
radical  which  has  not  yet  been  positively  identified  and  it  yields 
thy  mine  instead  of  uracil.  Levene  and  Jacobs  consider  that  thymus 
nucleic  acid  probably  possesses  the  following  structure: 

HOv 

O    =  PO—  C6HioO4—  C6H4N6O 
/  Guanine  group 

0/ 


HO 


O 


II 


PO—  C6H8O2—  C6H6N2O2 

Thymine  group 


O 


O    =  PO— CeHsO:!—  C4H4N3O 

Cytosine  group 


H 


O    =  PO— C6Hio04— C5H4NB 
/  Adenine  group 

HO/ 


184  NUCLEIC  ACIDS  AND  THE  NITROGENOUS  BASES 


AMINES  DERIVED  FROM  AMINO-ACIDS. 

The  proteolytic  enzymes,  such  as  Trypsin  and  Erepsin,  accomplish 
the  conversion,  by  hydrolysis,  of  the  proteins  into  their  constituent 
amino-acids.  The  next  step  in  the  degradation  of  nitrogenous  food- 
stuffs by  animal  tissues  generally,  appears  to  consist  in  Deaminization 
with  the  splitting  off  of  ammonia  and  the  oxidation  of  the  remainder 
of  the  original  amino-acid  molecule  to  carbon  dioxide  and  water.  No 
intermediate  stages  in  this  process  have  been  definitely  established, 
and  we  have  been  unable  to  detect  the  presence  in  animal  tissues  of 
enzymes  capable  of  producing  nitrogenous  bases  other  than  ammonia 
from  amino-acids.  That  such  enzymes,  perhaps  highly  localized,  do 
actually  exist  in  animal  tissues  may  be  regarded  as  exceedingly  prob- 
able, from  the  variety  and  physiological  importance  of  the  nitrogenous 
bases  which  are  found  to  occur  in  animal  tissues  and  their  significant 
chemical  resemblance  to  certain  of  the  amino-acids  which  are  yielded  by 
the  digestion  of  protein. 

Bacteria  and  other  Fungi,  however,  constitute  a  group  of  organisms 
which  are  able  to  rapidly  produce  from  amino-acids  a  series  of  nitrog- 
enous bases   which    arise   by   Decarboxylization   of    the    amino-acid 
molecule  in  accordance  with  the  general  equation: 
R 

-|    ,  R 

CHNH2 

=     CH2NH2     +     CO2 
COOH 

Amino-acid  Amine 

At  the  same  time  that  this  is  taking  place,  Deaminization  is  also 
proceeding,  and  is  evidenced  by  the  production  of  ammonia.  The 
conditions  determining  the  relative  proportion  of  these  two  processes 
are  complex  and  have  not  as  yet  been  fully  determined,  but  it  has 
been  observed  that  the  presence  of  carbohydrates  in  a  culture  of 
bacteria  or  fungi  greatly  diminishes  the  production  of  ammonia, 
presumably  because  in  the  absence  of  carbohydrates  the  organisms 
utilize  amino-acids  as  a  source  of  energy  as  well  as  a  source  of  nitro- 
gen, and  consuming  the  carbon  and  hydrogen  components  for  this 
purpose,  split  off  ammonia  as  a  by-product.  In  studying  the  decar- 
boxylization of  individual  amino-acids  it  has  been  found  that  the 
addition  of  Peptone  to  the  bacterial  culture  increases  the  yield  of 
amines,  probably  because  the  process  of  deaminization  being  shared 
between  the  amino-acid  and  the  peptone,  a  greater  proportion  of  the 
amino-acid  remains  available  for  decarboxylization. 

Decarboxylization  may  also,  especially  under  anaerobic  conditions, 
be  accompanied  by  reduction,  in  which  case  Formic  Acid  is  produced 
instead  of  carbon  dioxide: 
R 

I  R 

CHNH2  | 

+  H2     =     CH2NH2     +     HCOOH 
COOH 
Amino-acid  Amine 


AMINES  DERIVED  FROM  AMINO-ACIDS 


185 


The  greatest  importance  of  this  process  from  a  biochemical  point  of 
view  arises  out  of  the  intense  physiological  activity  of  many  of  the 
products  which  originate  in  this  manner,  the  resemblance  of  some  of 
these  products  to  the  active  principles  of  certain  of  the  glands  of 
internal  secretion,  and  from  the  probability  that  some  of  them  may 
reach  the  circulation,  occasionally  in  injurious  quantities,  by  absorp- 
tion from  the  large  intestine  wherein  they  are  produced  by  bacterial 
activity. 

The  following  amines  have  been  produced  from  the  corresponding 
amino-acids  by  the  action  of  putrefactive  bacteria.  It  is  possible, 
however,  that  the  true  source  of  methylamine  in  the  putrefaction 
of  fishes  is  not  glycocoll,  but  choline  (trimethyl  oxyethyl  ammonium 
hydroxide)  which  is  the  basic  constituent  of  Lecithin. 

Amino-acid.  Amine. 

Glycocoll,  CH2.NH2.COOH  Methylamine,  CH3NH2 

Alanine,  CH3.CH.NH2.COOH  Ethylamine,  CH3CH2NH2 


Valine, 


cH.CH(NH2).COOH 


Isobutylamine, 


CH.CH2NH2 


CH; 


\ 


Leucine, 


\ 


CH.CH2.CH(NH2).COOH    Isoamylamine,  ^>CH.CH2CH2.NH2 

CH/ 


CH 


3\ 


CH3v 


Isoleucine,  NcH.CH(NH2).COOH      Ethylmethyl  ethylamine    \CH.CH2NH2 

c2H6/  CZH/ 

Phenylalanine,  C6H5CH2.CH(NH2).COOH     Phenylethylamine,  C6H5.CH2.CH2.NH2 
Tyrosine,  HO.C6H4.CH2.CH(NH2).COOH      p-Hydroxyphenylethylamine    (tyramine), 

HO.C6H4.CH2CH2NH2 
,NH2 


Guanido-butylamine  (agmatine) , 

/NH2 
NH.CH2.CH2.CH2.- 

CH(NH2).COOH       HN    = 


Arginine,  HN 


^NH.CH2.CH2CH2CH2NH2 

Lysine,  H2N.CH2.CH2CH2.CH2.CH(NH2).-     Pentamethylene-diamine  (cadaverine), 

COOH  H2N.CH2CH2.CH2CH2.CH2NH2 

Ornithine,  H2N.CH2CH2.CH2.CH(NH2).-        Tetramethylene-diamine  (putrescine), 


COOH 

Histidine, 

CH 

/   \ 

N 


H2N.CH2.CH2.CH2.CH2NH2 

Iminazoylyl  ethylamine  (histamine,  erga- 
mine), 
CH 


NH 


CH  =  C.CH2.CH(NH2).COOH 
Tryptophane,  C.CH2.CH(NH2).COOH 

/\ 

C6H4  CH 

\/ 

NH 


N 


CH 


NH 


C.CH2.CH2.NH2 
Indolethylamine,  C.CH2CH2NH2 

/\ 

/         \ 

C6H4  CH 

\/ 

NH 


186          NUCLEIC  ACIDS  AND  THE  NITROGENOUS  BASES 

Pyrrolidine,  which  should  be  formed  by  decarboxylization  from 
proline,  oxypyrrolidine  which  should  be  formed  from  oxyproline, 
amino-ethyldisulphide  which  should  be  formed  from  cystine,  and 
/3-hydroxyethylamine  which  should  be  formed  from  serin,  have  not 
yet  been  found  possible  to  prepare  by  bacterial  decarboxylization. 

While  a  wide  variety  of  bacilli,  especially  anaerobes,  are  able  to 
bring  about  the  decarboxylization  of  amino-acids,  this  power  would 
seem  to  be  possessed  in  an  exceptional  degree  by  a  specific  organism, 
Bacillus  aminophilus  intestinalis  which  has  been  isolated  by  Bertrand 
and  Berthelot. 

The  production  of  these  bases,  many  of  which  are  definitely  toxic 
is  not  necessarily  accompanied  by  the  production  of  the  odor  which 
is  commonly  considered  to  be  indicative  of  putrefaction.  The  odor 
of  putrefaction  is  due  to  Indol  and  Skatol  or  /3-methyl  indol: 

CH  C.CH3 

/\  '        /\ 

C6H4        CH  C6H4        CH 

\/  .  \/ 

NH  NH 

Indol.  Skatol. 

and  these  substances  which  are  derived  from  Tryptophane  are  the 
products  of  a  further  stage  of  putrefactive  decomposition,  arising  by 
combined  decarboxylization  and  deaminization  succeeded  by  partial 
(skatol)  or  complete  (indol)  oxidation  of  the  aliphatic  hydrocarbon 
chain  of  the  tryptophane  molecule. 

The  bases  which  are  derived  in  this  way  from  the  proteins  dis- 
play the  usual  characteristic  properties  of  the  amines.  They  are  very 
much  more  basic  than  the  amino-acids  from  which  they  are  derived, 
and  yield  crystalline  salts  with  mineral  acids. 

The  aliphatic  monamines  (methylamine,  ethylamine,  isobutyl- 
amine,  isoamylamine,  dimethylaminobutane)  exert  a  physiological 
action  mimicking  the  effects  of  stimulation  of  the  sympathetic  nervous 
system,  they  are  therefore  termed  by  Barger  and  Dale  ''Sympathomi- 
metic"  bases.  The  lowest  amine  to  produce  a  distinct  rise  in  blood 
pressure  on  intravenous  injection  is,  however,  Isobutylamine ;  the 
activity  increases  with  increasing  length  of  the  aliphatic  hydrocarbon 
chain  up  to  Hexylamine,  and  thereafter  declines  as  the  number  of 
carbon  atoms  increases.  Very  much  more  effective  than  mere  increase 
in  the  length  of  the  chain  is,  however,  the  introduction  of  a  ring- 
structure  as  in  the  benzol  and  heterocyclic  derivatives.  Thus  Phenyl- 
ethylamine  is  at  least  five  times  as  active,  physiologically,  as  any 
aliphatic  amine.  Two  milligrammes  of  this  substance  when  injected 
intravenously  may  increase  the  blood-pressure  of  a  cat  no  less  than 
six  hundred  per  cent.  (30  mm.  to  180  mm.).  The  most  active,  however, 
of  the  monamines  derived  from  the  amino-acid  cleavage-products  of 
protein  is  parahydroxyphenylethylamine  (Tyramine)  which  exerts  an 


AMINES  DERIVED  FROM  AMINO-ACIDS  187 

effect  upon  blood-pressure  about  one-twentieth  of  that  exerted  by 
Adrenaline.  When  injected  intravenously  it  causes  a  rapid  and  pro- 
nounced rise  in  blood-pressure  which  is  somewhat  more  prolonged 
than  the  rise  which  is  caused  by  injections  of  adrenaline.  Unlike 
adrenaline,  howrever,  tyramine  does  not  cause  any  vasoconstriction 
when  applied  locally  to  mucous  surfaces,  and  large  doses  fail  to  produce 
the  glycosuria  which  results  from  adrenaline-poisoning.  Tyramine, 
furthermore,  has  a  decided  action  upon  the  uterus,  causing  the  non- 
pregnant  uterus  to  relax  while  the  pregnant  uterus  is  stimulated  to 
contraction.  The  glands  which  are  innervated  by  the  sympathetic 
system  are  stimulated  by  tyramine. 

It  has  been  considered  possible  that  since  tyramine  may  be  produced 
in  vitro  from  Tyrosine  by  the  action  of  fecal  bacteria,  the  presence  of 
this  substance  in  the  large  intestine  and  its  absorption  may  be  respons- 
ible for  pathological  conditions  in  which  high  blood-pressure  is  a 
leading  symptom.  As  in  the  case  of  adrenaline,  prolonged  adminis- 
tration of  tyramine  leads  to  renal  and  vascular  lesions  similar  to 
those  which  so  generally  accompany  persistent  arterial  hypertension 
in  man. 

Indolethylamine  is  not  so  potent  as  tyramine  and  differs  from  it 
in  several  details  of  its  action,  notably  in  giving  rise  to  muscular 
tremors  or  even  convulsions,  due  to  a  transient  stimulation  of  the 
central  nervous  system.  Indolethylamine  has  also  a  direct  stimula- 
tory action  on  smooth  muscle-fibers,  which  is  especially  marked  in  the 
arterioles  of  the  iris  and  the  uterus. 

Among  the  Diamines,  Putrescine  and  Cadaverine  are  of  historic 
interest  as  they  were  among  the  earliest  putrefaction-bases  to  be 
isolated,  definitely  characterized  and  identified.  They  are,  however, 
comparatively  innocuous  substances,  having  very  slight  physiological 
activity  and  in  common  with  other  diamines,  but  in  contrast  to  the 
monamines,  they  cause  a  fall  in  blood-pressure  when  they  are  injected 
intravenously.  They  occur  in  the  urine  in  cases  of  cystinuria,  their 
presence  indicating  a  defective  power  of  the  tissues  to  deaminize 
amino-acids. 

Agmatine  has  a  direct  action  upon  the  muscular  tissues  of  the 
uterus,  inducing  contractions;  it  is,  however,  very  much  less  potent 
in  this  respect  than  Ergamine  which,  with  Ergotoxine  and  Tyramine  is 
the  active  principle  of  the  pharmaceutical  preparations  of  ergot. 

Ergot  is  a  parasitic  fungus,  Claviceps  purpurea,  which  grows  on 
diseased  rye,  and  has  been  employed  from  very  ancient  times  to  cause 
contractions  of  the  uterus.  The  amines  which  it  contains  are  undoubt- 
edly produced  by  this  fungus,  as  they  are  by  other  fungi  and  bacteria, 
by  decarboxylization  of  the  corresponding  amino-acids.  Ergamine 
stimulates  unstriated  muscle-cells  directly,  inducing  especially  powerful 
contractions  of  the  uterus,  but  also  stimulating  smooth-muscle  fibers 
in  other  organs,  for  example  the  stomach  and  intestine  and  the  con- 
strictor muscles  of  the  pupil  of  the  eye.  When  dissolved  in  physio- 


188  NUCLEIC  ACIDS  AND  THE  NITROGENOUS  BASES 

logical  saline  solution  and  perfused  through  excised  bloodvessels  the 
muscle-fibers  of  the  vessels  contract,  causing  a  decrease  in  their 
diameter,  but  when  er gamine  is  injected  intravenously,  the  effect 
upon  the  majority  of  the  vessels  in  situ  is  just  the  reverse,  and  the 
blood-pressure  undergoes  a  profound  decrease  due  to  their  dilation. 
The  vessels  of  the  lungs,  heart  and  kidneys,  however,  are  constricted. 
An  exceptionally  interesting  action  of  ergamine  is  that  of  inducing 
spasmodic  contractions  of  the  Bronchioles  when  administered  in 
relatively  large  doses.  Thus  0.5  milligrams  of  ergamine  intra- 
venously injected  will  kill  a  guinea-pig  in  a  few  minutes,  and  the 
cause  of  death  is  asphyxiation,  which  is  due  to  closure  of  the  bron- 
chioles, preventing  the  passage  of  air  into  or  out  of  the  lungs.  Post- 
mortem examination  shows  that  the  lungs  are  permanently  dilated 
(Emphysema).  Now  this  is  the  condition  which,  in  a  milder  degree, 
is  responsible,  in  human  beings,  for  the  respiratory  distress  in  Asthma. 
It  may  further  be  brought  about  by  peptone-poisoning  or  by  inducing 
Anaphylactic  Shock. 

When  a  non-toxic  foreign  protein,  for  example  egg-white,  is  injected 
hypodermically  or  into  the  circulation  of  an  animal,  if  the  first  dose  is 
followed  within  a  few  days  by  a  second,  that  in  a  like  period  by  a 
third,  and  so  forth,  no  harmful  results  ensue,  and  the  animal  gradually 
acquires  Immunity  to  the  protein.  If,  however,  after  the  injection 
of  the  first  dose  of  protein  a  considerable  period,  e.  g.,  three  weeks,  be 
allowed  to  elapse  before  the  second  is  administered,  if  the  second 
dose  be  sufficiently  large,  a  condition  of  "  anaphylactic  shock"  is 
induced  which  is  frequently  fatal.  The  cause  of  death  is  asphyxiation 
due  to  spasmodic  contractions  of  the  bronchioles  and  it  is  believed  that 
the  preliminary  "  sensitization"  of  the  animal  has  endowed  its  tissues 
with  the  ability  to  so  rapidly  decompose  the  foreign  protein  that  upon 
injection  of  the  second  dose  dangerous  quantities  of  toxic  peptones  or 
other  products  of  protein  decomposition  are  rapidly  formed.  The 
resemblance  between  the  symptoms  of  ergamine  poisoning,  peptone 
poisoning,  asthma  and  anaphylactic  shock  is  so  striking  as  to  suggest 
a  common  cause  and  the  view  has  been  advanced  that  all  of  these 
phenomena  are  attributable  to  the  liberation  of  j8-iminazolyl  ethyl- 
amine  in  the  blood  or  tissues,  the  source  of  the  substance  being  the 
Histidine  radical  in  proteins  or  peptones.  On  the  other  hand  it  has 
not  been  conclusively  shown  that  peptones  themselves  or  peptide- 
derivatives  of  /3-iminazolyl  ethylamine  may  not  produce  like  effects. 
At  any  rate  a  part  of  the  symptoms  of  anaphylactic  shock  are  not 
attributable  to  ergamine,  because  this  substance  does  not  render 
blood  incoagulable,  while  incoagulability  of  the  blood  is  one  of  the 
symptoms  of  profound  anaphylactic  shock  and  of  peptone  poisoning. 

The  possibility  of  the  formation  of  /3-iminazolyl  ethylamine  from 
proteins  in  the  lower  intestine  by  the  action  of  fecal  bacteria  may 
enable  us  to  trace  certain  forms  of  asthma  to  an  intestinal  source. 
The  majority  of  cases  appear,  however,  to  be  undoubtedly  anaphy- 


THE  BETAINES  AND  THE  VITAMINES  189 

lactic,  the  immediate  origin  of  an  attack  being  frequently  traceable  to 
ingestion  of  some  protein  to  which  the  individual  in  question  has 
become  sensitized,  e.  g.,  the  proteins  in  the  sweat  of  horses,  egg-white, 
the  proteins  in  strawberries  or  in  pollen,  or  possibly  proteins  pro- 
duced locally  by  bacterial  infections.  On  the  other  hand  asthmatic 
attacks,  originally  anaphylactic,  may  frequently  be  seen  in  early 
cases  to  pass  through  transitional  stages  into  habitual  reflexes,  which 
are  thereafter  elicited  by  any  unusual  stimulus,  e.  g.,  emotional  excite- 
ment or  indigestion.  The  problem  is  therefore  a  many-sided  one  of 
which  the  several  factors  are  frequently  difficult  or  impossible  to 
disentangle. 

Closely  related  to  the  amines  which  we  have  been  considering  are 
the  co-Amino-acids  in  which  the  a-amino-group  which  is  so  characteristic 
of  the  amino-acids  derived  from  proteins  is  absent,  the  ami  no-group 
being  attached  to  a  carbon  atom  which  is  remote  from  the  carboxyl- 
group.  This  results  in  greatly  increased  basicity  of  the  amino-acid 
so  that  these  compounds  resemble  the  amines  in  chemical  behavior 
rather  than  the  amino-acids.  They  may  be  produced  in  putrefaction 
by  partial  deaminization  of  a  diamino-acid,  as  in  the  production  of 
6-Amino-valeric  Acid  from  ornithine: 

H2N.CH2.CH2.CH2.CH(NH2)COOH  +  2H  =  H2N.CH2.CH2.CH2.CH2.COOH  +  NH3 

or  they  may  result  from  partial  decarboxylization  of  a  dicarboxylic 
acid,  as  in  the  production  of  7-Aminobutyric  Acid,  from  glutamic  acid: 

HOOC.CH(NH2).CH2.CH2.COOH  =  H2N.CH2.CH2.CH2.COOH  +  CO2 

An  important  representative  of  this  group  of  substances  is  Carnosine 
which,  next  to  creatine,  is  the  most  abundant  nitrogenous  base  in 
meat-extracts.  It  is  present  in  horse-meat  to  the  extent  of  1.8  grams 
per  kilo.  On  hydrolysis  it  yields  Histidine  and  /5-Alanine  in  equi- 
molecular  proportions.  It  is  believed  to  be  a  dipeptide  histidyl-/3- 
alanine: 

CH  —  =  C—  CH2.CH.COHN.CH2.CH2COOH 

I 

N  NH         NH2 


THE  BETAINES  AND  THE  VITAMINES. 

The  Betaines  are  amino-acids  in  which  the  nitrogen  atom  is  united 
to  methyl-groups  in  the  place  of  hydrogen  atoms.  These  substances 
in  the  absence  of  water,  form  cyclic  anhydrides  which  open  up  when 
they  are  dissolved  in  water  or  unite  with  acids.  Thus  Betaine  itself, 
or  Trimethylglycine  has  the  formula: 

o 


(CH8)3N< 

CH2 


190          NUCLEIC  ACIDS  AND  THE  NITROGENOUS  BASES 

When  dried  at  above  100°;   but  when  it  is  dissolved  in  water  or 
combined  with  acids  it  is  probably  represented  by  the  formula : 

OH 


Betaine  occurs  in  the  sap  of  the  sugar-beet,  Beta  vulgaris,  and  is 
extracted  together  with  the  sugar,  remaining  in  the  molasses  when  the 
sugar  is  refined.  It  is  non-toxic  and  is  not  utilizable  by  animals  as 
a  food,  but  it  is  stated  that  the  creatine  content  of  the  muscles  is  per- 
ceptibly increased  by  administration  of  betaine. 

Trimethyl  Histidine  is  found  in  edible  mushrooms.  The  constitu- 
tion is: 

CH-  x 

CH 


The  corresponding  betaine  of  tryptophane  is  Hypaphorine.  Up  to 
the  present  these  betaines  have  only  been  found  in  plant-tissues. 

In  putrefying  meat  we  find  a  betaine  which  unlike  those  described 
above,  has  a  powerful  physiological  action.  This  is  7-n-butyro-betaine, 
the  betaine  of  7-amino-butyric  acid : 


It  has  an  action  upon  nerve-endings  resembling  that  of  curare  and 
when  injected  produces  convulsions,  dyspnea  and  paralysis. 
Carnitine  is  the  a-hydroxy  derivative  of  7-butyro-betaine : 


it  is  found  in  meat-extracts  and  is  almost  devoid  of  immediate  physio- 
logical actions. 


THE  BETAINES  AND  THE  VITAMINES  191 

Trigonelline  is  the  betaine  of  nicotinic  acid  and,  therefore,  unlike  the 
betaines  heretofore  considered,  is  not  obtainable  from  any  amino-acid 
cleavage-product  of  proteins.  Its  constitution  is  as  follows: 

/CH\ 


\ 

HC  C.CO 


HC  CH 


I 
CH3 

it  is  devoid  of  any  obvious  physiological  action,  but  is  of  especial 
interest  because  in  the  first  place  of  its  wide  distribution  in  a  variety 
of  vegetable  tissues  and  in  the  second  place  because  Nicotinic  Acid, 
from  which  it  is  derived  by  methylation,  occurs  in  the  polishings  from 
rice. 

In  various  parts  of  the  Orient,  but  particularly  Japan  and  the 
Philippines,  where  rice  constitutes  a  very  large  proportion  of  the 
dietary,  the  introduction  of  milling  methods  which  involve  stripping 
off  the  pericarp,  or  "polishing"  of  rice  has  led  to  the  widespread  occur- 
rence of  a  disease  known  as  Beri-Beri,  the  ravages  of  which  were  par- 
ticularly prominent  in  the  Japanese  army  during  the  Russo-Japanese 
War.  The  disease  is  evidenced  by  general  lassitude  accompanied  by 
anesthesia  in  certain  areas  of  the  skin,  edema  of  the  ankles  and  face, 
partial  paralysis  of  the  leg-muscles  and,  toward  the  termination  of  the 
disease,  distress  in  breathing.  These  symptoms  are  traceable  to  a 
widespread  peripheral  neuritis,  beginning  in  the  nerve-fibers  most 
remote  from  the  central  nervous  system  and  travelling  centripetally. 
The  mortality  is  very  high.  It  was  pointed  out  by  Eijkman  in  1897 
that  beri-beri  could  be  prevented  by  eating  unpolished  rice  with  the 
pericarp  intact,  and  that  it  could  furthermore  be  cured  by  the  adminis- 
tration of  rice-polishings  ("rice-bran").  He  discovered  that  a  very 
similar  disease,  involving  peripheral  polyneuritis  and  ultimate  death, 
could  be  induced  artificially  in  pigeons  by  feeding  them  exclusively  upon 
polished  rice.  The  inference  was  plain  that  a  preventive  and  curative 
substance  is  present  in  the  pericarp  of  rice. 

The  nature  of  this  substance  has  been  extensively  investigated  by 
C.  Funk  and  many  others.  Funk  has  succeeded  in  isolating  a  curative 
crystalline  substance  from  Yeast  which  is  exceedingly  potent,  as  little 
as  two  milligrammes  restoring  the  power  of  movement  within  three 
hours  to  pigeons  which  have  been  completely  paralysed  by  a  diet  of 
polished  rice.  Curative  substances  are  also  found  in  a  variety  of  other 
foodstuffs  and  in  animal  tissues.  They  are  soluble  in  water  and  in 
alcohol,  but  insoluble,  or  sparingly  soluble  in  ether.  An  active  curative 
substance  is  invariably  found  to  yield  a  blue  color  when  mixed  with 
Folin  and  Macallum's  "Uric  Acid  Reagent,"  which  is  a  solution  of 
sodium  phosphotungstate  containing  a  specified  proportion  of  phos- 


192          NUCLEIC  ACIDS  AND  THE  NITROGENOUS  BASES 

phoric  acid  and  sodium  tungstate.  A  similar  color  is  yielded  by  uric 
acid,  alloxantin,  dihydrophenylalanine,  amino-tyrosine,  and  certain 
di-  or  tri-phenols,  but  not  by  purine  or  pyrimidine  bases  other  than 
those  mentioned,  nor  by  tyrosine  itself. 

The  curative  substances  isolated  by  Funk  have  been  termed  by  him 
Vitamines.  From  rice-polishings  a  crystalline  curative  fraction  was 
obtained  which,  on  fractional  crystallization  was  separated  into  two 
substances.  The  one  proved  to  be  Nicotinic  Acid,  which,  in  the  pure 
crystallized  condition,  is  devoid  of  curative  action,  and  the  other  an 
unidentified  nitrogenous  substance  which  tends  to  lose  its  curative 
power  with  successive  purifications.  The  curative  substance  from 
yeast  was  similarly  found  to  yield  nicotinic  acid  and  an  unknown 
nitrogenous  base. 

From  the  fact  that  some  of  the  pyrimidine  derivatives  have  a  weak 
curative  action  on  polyneuritis,  it  was  at  first  thought  that  the  vita- 
mines  were  probably  pyrimidine  derivatives.  The  more  recent 
investigations  of  R.  R.  Williams  indicate  that  the  curative  principles 
may  be  substances  having  a  Betaine  structure.  Thus  a-Hydroxy- 
pyridine  has  a  definitely  curative  action  upon  artificially  induced  poly- 
neuritis,  so  long  as  it  yields  needle-shaped  crystals,  but  these  crystals 
spontaneously  change,  on  standing,  into  crystalline  granules  which  are 
quite  devoid  of  antineuritic  properties.  Now  a-hydroxypyridine  may 
conceivably  exist  in  a  variety  of  chemical  forms  of  which  the  following 
are  examples : 

:H\  /CH\ 

\  /  \ 

HC  CH  HC  CH 


HC  Cx  HC  COH 


The  curative  variety,  yielding  needle-shaped  crystals,  is  probably 
the  pseudobetaine  form,  resembling  the  betaines  in  containing  the 
group: 


_N O 

I 

and  the  fact  that  this  structure  and  the  antineuritic  properties  of 
a-hydroxypyridine  spontaneously  disappear  on  standing  is  suggestive 
in  view  of  the  fact  that  the  curative  substances  isolated  from  yeast 
and  rice-polishings  by  Funk  tend  also  to  lose  antineuritic  power 
spontaneously  on  standing  or  on  repeated  purification. 

The  betaines  themselves,  such  as  trimethylglycine  or  trigonellin 
are  impotent  to  protect  pigeons  fed  on  polished  rice  from  the  develop- 
ment of  polyneuritis.  It  is,  however,  characteristic  of  the  betaines 
that  the  anhydride  ring  is  very  unstable  and  readily  opens  up,  as,  for 


THE  BETAINES  AND  THE  VITAMINES  193 

example,  when  salts  are  formed  with  acids,  and  failure  to  obtain 
marked  curative  results  with  these  substances  may  therefore  be  attrib- 
utable to  absence  of  the  above  ring-structure  in  the  preparations 
administered. 

The  antineuritic  substances  in  an  aqueous  extract  of  yeast  may  be 
removed  therefrom  by  shaking  up  the  fluid  with  fuller's  earth.  The 
fuller's  earth  then  becomes  "activated"  and  carries  with  it  all  the 
curative  substances.  An  alkaline  extract  of  this  activated  fuller's 
earth  was  found  by  Williams  and  Seidell  to  exert  a  marked  curative 
effect,  but  on  recrystallization  the  substance  lost  its  antineuritic 
properties  and  then  was  identified  as  Adenine.  On  heating  to  180° 
in  sealed  tubes  with  alcohol,  a  portion  of  the  antineuritic  activity  was 
regained,  and  at  the  same  time  it  acquired  the  power,  which  adeniue 
does  not  possess,  of  yielding  a  blue  color  with  Folin  and  Macallums' 
"uric  acid  reagent."  Williams  and  Seidell  infer  that  the  curative 
substances  in  this  instance  is  an  isomeric  modification  of  adenine. 

The  instability  of  the  curative  substances  and  the  minute  propor- 
tions in  which  they  are  present  in  antineuritic  foodstuffs  renders  the 
attainment  of  any  definite  conclusions  a  matter  of  exceptional  difficulty. 
In  the  meantime,  however,  and  pending  more  exact  knowledge  of  this 
subject,  very  great  care  should  be  taken  to  avoid  confusion  by  grouping 
together  essentially  dissimilar  substances  of  widely  differing  physio- 
logical significance  as  "vitamines."  Such  procedure  can  only  lead  to 
mystification,  obscures  the  issue,  and  obstructs  the  progress  of  our 
knowledge.  The  term  "vitamine"  should  be  definitely  restricted  to 
those  nitrogenous  substances  which  are  known  to  possess  curative 
action  upon  Polyneuritis.  While  a  variety  of  other  substances  are  now 
known  to  exist,  which,  in  relatively  small  amounts  are  essential  to 
health  or  growth,  yet  to  group  them  all  together  as  "vitamines" 
simply  deprives  the  name  of  its  scientific  significance.  It  is  much 
better  to  use  the  descriptive  term  "Accessory  Foodstuffs,"  invented  by 
their  discoverer,  Gowland  Hopkins,  to  include  all  dietetic  factors  which 
are  essential  constituents  of  the  diet  for  purposes  other  than  the 
provision  of  heat-units  or  the  building-up  of  carbohydrates,  fats  and 
proteins.  The  hydroxy-acids  and  other  substances  in  fruits  and 
vegetables  which  act  as  Antiscorbutics  or  preventives  of  scurvy  are 
therefore  "  accessory  foodstuffs"  but  they  are  not  vitamines.  We  shall 
make  further  reference  to  the  various  classes  of  accessory  foodstuffs 
in  later  chapters. 

Another  deficiency-disease  which  probably  depends  upon  lack  of 
vitamines,  or  of  substances  resembling  those  which  are  lacking  in 
polished  rice,  is  Pellagra,  a  condition  which  is  very  common  in  districts 
such  as  the  Southern  United  States,  where  maize  furnishes  a  large 
proportion  of  the  diet.  Milling  methods  which  involve  total  removal 
of  the  pericarp  of  the  grain  are  believed  to  be  responsible  for  the 
disease.  Maize  deprived  of  its  outer  covering  has  been  shown  to  cause 
polyneuritis  in  pigeons  in  the  same  way  as  polished  rice. 
1.3 


194         NUCLEIC  ACIDS  AND  THE  NITROGENOUS  BASES 

NITROGENOUS  BASES  DERIVED  FROM  GUANIDINE. 
Guanidine 

/NH2 
HN    =  C< 

XNH2 

is  obtained  from  proteins  by  the  employment  of  strong  oxidizing  rea- 
gents, its  presence  has  also  been  detected  in  various  vegetable  tissues, 
among  others  in  the  sugar-beet.  It  is  a  strong  base,  yielding  strongly 
alkaline  solutions  of  very  stable  salts  with  acids.  It  is  uncertain 
whether  or  not  it  occurs  in  traces  in  the  blood  and  tissues.  Methyl- 
guanidine 

/NH.CHa 
HN    =  C< 

XNH2 

is,  however,  a  normal  constituent  of  blood,  muscular  tissues  and  urine. 

Guanidine  and  methylguanidine  have  a  very  decided  physiological 
action,  two  hundred  milligrammes  of  methylguanidine  being  a  lethal 
dose  for  a  guinea-pig.  The  amount  of  methylguanidine  in  the  urine  is 
greatly  increased  by  Anaphylactic  Shock,  but  the  symptoms  of  poisoning 
are  nowise  similar  to  those  of  anaphylactic  shock.  They  consist  in 
fibrillar  twitchings  of  the  peripheral  muscles  and  an  excitation  of  the 
spinal  cord  resembling  in  comparatively  slight  measure  that  produced 
by  strychnine  or  by  Curare  when  directly  applied  to  the  cord.  In 
larger  doses  the  myoneural  junctions  are  paralyzed  in  the  same  way  that 
they  are  by  curare  and  the  spinal  centers  are  depressed.  The  fibrillar 
twitchings  produced  in  muscles  by  small  doses  of  guanidine  or  methyl- 
guanidine are  suppressed  by  calcium  salts  and  in  this  respect  as  well  as 
in  the  character  of  the  muscular  excitation,  the  action  of  small  doses 
of  guanidine  resembles  the  action  of  sodium  salts  upon  nerves  and 
muscles. 

The  marked  effect  of  methylguanidine  upon  neuromuscular  tissues 
is  of  especial  interest  because  a  derivative  of  methylguanidine,  Creatine, 
or  methyl-guanidine-acetic  acid: 

/N.CH3.CH2.COOH 
HN    =  C< 

XNH2 

is  the  most  abundant  nitrogenous  base  in  muscular  tissues.  The  per- 
centage of  creatine  varies  in  different  muscles,  being  higher  in  voluntary 
(striated)  than  in  involuntary  (smooth)  muscles.  In  given  muscles 
the  percentage  of  creatine  varies  in  different  species  of  animals,  but  is 
remarkably  constant  in  different  individuals  of  the  same  species.  The 
following  are  the  percentages  of  creatine  found  in  the  muscle  of  various 
animals  by  Myers  and  Fine. 

Per  cent. 
Species.  of  creatine. 

Rabbit 0.52 

Cat 0.45 

Man 0.39 

Dog .     a.  37 


NITROGENOUS  BASES  DERIVED  FROM  GUANIDINE        195 

In  the  urine  the  anhydride  of  creatine,  Creatinine,  is  an  important 
constituent: 

/N.CH3.CH2.COOH  /N.CHs.CHs 

HN    =  C<                                                  HN    =  C<  I  +     H2O 

XNH2  XNH CO 

As  a  rule  creatine  itself  is  not  found  in  mammalian  urine,  although 
it  replaces  creatinine  in  the  urine  of  birds  and  is  a  normal  constituent 
of  the  urine  of  young  children.  In  women  creatine  occurs  in  the  urine 
immediately  after  menstruation  and  occurs  in  large  amounts  in  the 
urine  during  the  involution  of  the  uterus  which  follows  delivery.  It  is 
considered  probable  by  Folin  and  others  that  the  creatinine  in  urine  is 
not  derived  from  the  creatine  of  the  muscles  but  represents  a  product 
of  the  catabolism  of  protoplasm.  Creatine  administered  by  mouth  in 
small  doses  does  not  appear  in  the  urine  either  as  such  or  as  creatinine. 

Neither  urinary  creatinine  nor  the  creatine  in  muscles  is  increased 
by  muscular  work,  but  the  creatine  content  of  muscles  appears  to  be 
connected  with  their  Tonus  or  degree  of  moderate  contraction  when  at 
rest.  Thus  standing  at  "attention"  in  a  military  position  increases 
the  urinary  creatinine  while  a  long  march  does  not.  On  the  other 
hand  if,  as  much  of  the  evidence  seems  to  indicate,  urinary  creatinine 
is  not  derived  from  the  creatine  of  muscles  but  from  the  "wear  and 
tear",  of  tissues  this  result  may  merely  indicate  that  standing  at 
"attention"  involves  more  destruction  of  muscular  tissues  than  the 
performance  of  muscular  work. 

Creatine  is  one  of  the  relatively  few  substances  which  stimulate  the 
gray  matter  or  Neurones  of  the  cerebral  cortex.  The  customary 
stimulants  for  nerve-fibers,  calcium-precipitating  substances,  barium 
chloride  and  so  forth,  have  no  action  upon  nerve-cells.  Creatine  is 
devoid  of  stimulating  action  upon  nerve-cells  but  when  applied  to  the 
motor  areas  of  the  cortex,  it  throws  the  animal  into  convulsions.  This 
may  be  connected  with  the  fact  that  the  convulsions  which  accompany 
Eclampsia,  a  metabolic  disease  of  pregnancy,  are  heralded  by  a  sharp 
rise  in  the  creatine  output  in  the  urine. 

Creatinine  may  be  detected  by  Jaffe's  Reaction,  which  consists  in  the 
red  color  produced  by  creatinine  in  alkaline  solutions  when  Picric  Acid 
is  added.  The  color  is  due  to  Picramic  Acid  which  is  formed  by  reduc- 
tion of  picric  acid.  This  reaction  is  employed  for  the  quantitative 
estimation  of  creatinine.  Creatinine  also  gives  a  ruby-red  color  with 
Sodium  Nitroprusside  in  alkaline  solution  (Weyl's  Reaction). 

Creatine  may  be  converted  into  creatinine  by  boiling  with  dilute 
hydrochloric  acid,  or  it  may  be  determined  directly  by  utilizing  the 
pink  coloration  which  it  yields  with  Diacetyl. 

It  should  be  noted  that  creatine  is  closely  related  to  Arginine,  which 
is  the  only  product  of  protein  hydrolysis  that  contains  a  guanidine 
radical : 

/CH3 

/NH.CH2.CH2.CH2.CH(NH2)COOH  /N< 

HN    =  C<  HN    =  C<      XCH2COOH 
XNH2  XNH2 

Arginine.  Creatine. 


196         NUCLEIC  ACIDS  AND  THE  NITROGENOUS  BASES 


THE  NITROGENOUS  BASES  DERIVED  FROM  THE  PHOSPHOLIPINS. 

The  saponification  of  the  Lecithins  by  alkalies  yields,  besides  soaps 
and  the  glycerophosphate  of  the  alkali,  a  nitrogenous  base,  Choline 
or  trimethyloxyethylammonium  hydroxide. 

/CH2.CH2OH 

(CH3)3  i  N< 

XOH 

It  is  a  strong  base,  yielding  alkaline  solutions  and  forming  a  double 
salt  with  platinic  chloride.  By  the  saponification  of  Kephalins,  how- 
ever, we  obtain  a  different  base,  namely  Amino-ethyl  Alcohol. 

H2N.CH2.CH2OH 

from  which  choline  is  probably  derived  by  methylation. 

There  has  been  much  discussion  of  the  question  whether  or  not 
a  third  and  related  base,  Neurine,  or  vinyltrimethyl  ammonium 

hydroxide : 

/CH:CH2 

*  (CH3)3  i  N< 

XOH 

is  yielded  by  the  hydrolysis  of  Protagon,  but  the  consensus  of  opinion 
appears  now  to  coincide  with  the  view  originally  expressed  by  Gule- 
witsch,  that  neurine  is  in  reality  a  putrefaction-product  derived  from 
choline  by  the  action  of  bacteria.  Thus  perfectly  fresh  brain-tissue 
does  not  appear  to  yield  neurine  at  all,  unless  the  lecithins  (or  protagon) 
are  boiled  with  strong  alkalies  which,  even  in  pure  solutions,  results  in 
a  partial  conversion  of  choline  into  neurine. 

Both  choline  and  neurine  exert  the  physiological  actions  which  are 
typical  of  all  the  trimethylamine  derivatives.  The  first  symptom  of 
poisoning  is  salivation,  followed  by  intestinal  cramps.  There  is  a 
preliminary  fall  in  blood- pressure  succeeded  by  a  rise.  Death  is 
ultimately  due  to  arrest  of  the  heart.  These  symptoms  arise  from 
stimulation  of  sympathetic  nerve-endings  in  the  glands  or  muscles 
affected  and  are  prevented  by  the  administration  of  Atropine  which 
paralyzes  these  junctions. 

It  was  at  one  time  thought  that  free  choline  might  occur  in  the  brain, 
particularly  in  degenerative  changes  of  the  central  nervous  system, 
and  that  under  these  conditions  choline  might  be  found  in  the  cerebro- 
spinal  fluid.  The  presence  of  choline  in  cerebrospinal  fluid  was,  in 
fact,  suggested  as  a  means  of  detecting  degenerative  lesions  of  the 
brain.  Since  platinic  chloride  must  be  employed  to  detect  the  small 
quantities  of  choline  looked  for,  however,  and  potassium  and  ammonium 
salts,  both  of  which  are  also  present,  yield  very  similar  crystalline 
platinichlorides,  it  is  rather  probable  that  the  crystals  obtained  from 
cerebrospinal  fluid  are  not  in  reality  compounds  of  choline.  The 


ACTIVE  PRINCIPLES  OF  INTERNAL  SECRETIONS          197 

small  quantities  which  are  obtainable  renders  investigation  of  this 
question  by  direct  analysis  a  very  difficult  one.  The  free  choline 
alleged  to  have  been  detected  in  brain-tissue  has  been  found  to  be  a 
postmortem  product  arising  from  autolysis  or  putrefaction. 

The  physiological  action  of  neurine  is  much  more  intense  than  that 
of  choline,  being  effective  in  about  one  twentieth  of  the  dosage.  By 
introducing  radicals  into  the  oxyethyl  group,  however,  as  in  Acetyl- 
choline  and  the  nitrous  acid  or  nitric  acid  esters  of  choline,  substances 
of  very  much  more  intense  physiological  activity  than  choline  itself 
are  produced. 

By  the  hydrolysis  of  the  Cerebrosides,  phrenosin  and  kerasin,  a 
nitrogenous  base,  Sphingosine  is  obtained  the  constitution  of  which  is 
at  present  unknown.  Its  percentage  composition  corresponds  to  the 
formula  Ci7H35NO2,  and  it  is  a  diatomic  alcohol  containing  an  amino- 
group.  When  sphingosine  is  heated  with  concentrated  sulphuric  acid 
and  a  sugar,  it  yields  a  purple- violet  coloration.  The  cerebrosides, 
when  similarly  treated,  first  dissolve  in  the  sulphuric  acid,  yielding  a 
clear  yellow  solution,  and  then  the  sphingosine  is  split  off  and  sepa- 
rates out  in  droplets  which  yield  the  reaction.  The  addition  of  sugar 
in  this  case  is  unnecessary  because  it  is  supplied  by  the  galactose  in 
the  cerebroside.  Regarding  the  physiological  actions  of  sphingosine 
nothing  definite  is  known. 

NITROGENOUS   BASES   FORMING   THE   ACTIVE  PRINCIPLES   OF 
INTERNAL  SECRETIONS. 

Of  these  the  best  studied  and,  therefore,  the  best  known  is  Adrenaline, 
the  blood-pressure  raising  or  Pressor  principle  of  the  Suprarenal  Gland. 
Other  names  by  which  this  substance  is  designated  in  current  literature 
are  Epinephrin,  Adrenin  and  Suprarenin.  The  term  adrenaline  is  that 
most  customarily  used  although  epinephrin  is  also  frequently  employed. 

Adrenaline  is  a  derivative  of  Catechol  and  possesses  the  following 
constitutional  formula : 

COH 

/\ 

HC  COH 


HC  CH 

\/ 

C 
CHOH 

CH2.NH.CH3 

Adrenaline. 


198         NUCLEIC  ACIDS  AND  THE  NITROGENOUS  BASES 

it  is  therefore,  a  methylamine  and  also  a  dihydroxybenzene  derivative. 
The  structure  of  adrenaline  may  be  compared  with  that  of  tyrosine: 

COH 

/\ 

HC  CH 


HC  CH 

\/  : 

c 

CH2 

CH(NH2)COOH 
Tyrosine. 

from  which  it  will  be  evident  that  adrenaline  and  tyrosine  contain  the 
same  skeleton  of  carbon  atoms. 

The  immense  physiological  importance  of  the  suprarenal  gland  was 
first  demonstrated  by  Addison,  in  1849,  when  he  showed  that  the 
disease  now  named  after  him,  was  connected  with  degenerative  changes 
of  the  suprarenal  bodies.  A  few  years  later  it  was  also  discovered 
that  the  suprarenal  glands  contain  a  "  chromogenic  substance"  which 
yields  a  vivid  green  color  with  ferric  chloride  and  a  red  color  with 
iodine.  Strangely  enough,  however,  the  remarkable  effect  of  suprarenal 
extracts  upon  blood-pressure  was  not  discovered  until  1894,  and  the 
positive  identification  of  the  pressor-substances  with  the  "  chromogenic 
substance"  was  not  established  until  some  years  later. 

Pure  adrenaline  crystallizes  in  colorless  spherules;  it  is  sparingly 
soluble  in  water  and  almost  insoluble  in  most  organic  solvents,  it  will 
however,  dissolve  in  glacial  acetic  acid,  ethyl  oxalate  or  benzaldehyde. 

Adrenaline  may  be  prepared  from  fresh  suprarenal  glands  by  extract- 
ing the  minced  tissue  with  water,  coagulating  the  proteins  by  heat  or 
trichloracetic  acid,  concentrating  the  extract  and  adding  ammonia 
which  causes  the  adrenaline  to  separate  out. 

The  readily  oxidizable  catechol  (orthodihydrobenzol)  nucleus  in 
adrenaline  is  responsible  for  a  variety  of  color  reactions  which  it  yields. 
The  following  are  among  the  most  characteristic: 

Ferric  Chloride  Reaction. — In  neutral  or  faintly  acid  solutions  adrena- 
line yields  with  ferric  chloride  a  vivid  grass-green  color,  which  changes 
to  violet,  reddish  violet  and  red  on  rendering  the  solution  alkaline. 
This  will  detect  about  one  part  of  adrenaline  in  thirty  thousand,  but 
the  addition  of  Sulphanilic  Acid,  while  changing  the  color  to  reddish 
brown,  also  renders  the  test  much^more  sensitive. 

Iodine  Reaction. — With  iodine  or  iodic  acid  adrenaline  yields  a  red 
color.  The  excess  of  iodine  may  be  removed  by  shaking  up  the  mixture 
with  ether. 

Mercuric  Chloride. — With  mercuric  chloride,  in  the  presence  of 
Calcium  Salts,  which  act  as  catalyzers,  solutions  of  adrenaline  yield  a 
red  color. 


ACTIVE  PRINCIPLES  OF  INTERNAL  SECRETIONS    190 

Persulphate  Reaction. — This  is  the  most  delicate  of  all  the  tests  for 
adrenaline,  detecting  one  part  of  adrenaline  in  five  million  of  solution. 
Potassium  persulphate  is  added  to  the  solution  to  the  extent  of  one- 
tenth  of  a  per  cent.,  and  the  test-tube  is  then  heated  by  immersion  in 
boiling  water. 

Phosphotungstic  Acid  Reaction. — With  Folin  and  Macallum's  "uric 
acid  reagent,"  which  consists  of  a  mixture  in  specified  proportions  of 
sodium  tungstate  and  phosphoric  acid,  adrenaline  yields  the  blue  color 
which  Uric  Acid,  Alloxantin,  certain  Dihydrophenols,  Aminotyrosine  and 
other  substances  including  the  vitamines  also  yield.  The  test  will 
detect  one  part  in  3  million  of  adrenaline. 

Adrenaline  constitutes  about  one-tenth  of  a  per  cent,  of  the  fresh 
tissue  of  the  suprarenal  gland.  One  bullock's  gland,  weighing  about 
ten  grams,  should  therefore  yield  about  ten  milligrams. 

The  origin  of  adrenaline  is  unknown.  The  close  relationship  to 
Tyrosine  would  suggest  this  as  the  parent  substance,  but  the  trans- 


Normal 


A 


20  mg.Tethelin     20  mg.Tethelin    1  cc.  Split  Product 


-*—  Minutes  — > 
FIG.  6. — Tracing  showing  effect  on  the  uterus  (guinea-pig)  of  split  products  of  tethelin. 

formation  of  tyrosine  into  adrenaline  would  involve  a  series  of  changes 
not  merely  decarboxylization,  but  also  the  introduction  of  two  hydroxyl- 
groups,  one  of  them  in  the  benzol-ring,  followed  by  methylation 
of  the  resultant  compound.  We  are  not  familiar  with  any  mechanism 
which  could  bring  about  this  rather  complicated  series  of  transforma- 
tions. There  are  some  indications,  however,  that  the  suprarenal 
glands  may  contain  precursors  of  adrenaline  which  are  devoid  of 
pressor-action,  and  yet  yield  a  coloration  with  oxidizing-agents. 

The  important  physiological  actions  of  adrenaline  will  be  separately 
discussed  (cf.  Chapter  XVI). 

The  posterior  lobe  or  Infundibulum  of  the  Pituitary  Body  contains  a 
nitrogenous  substance  which  exerts  an  action  upon  the  uterus  as 
distinctive  as  that  of  ergot  and  has  also  the  peculiar  property  of  exciting 
the  secretion  of  the  Mammary  Glands.  The  structure  and  even  the 
composition  of  the  active  substance  are  unknown,  but  since  it  yields  a 
red  color  with  Diazobenzene  Sulphonic  Acid  (see  Histidine),  it  probably 


200         NUCLEIC  ACIDS  AND  THE  NITROGENOUS  BASES 

contains  an  iminazolyl  radical  and  is,  therefore,  related  to  Ergamine. 
The  active  substance  which,  in  aqueous  solutions,  is  known  by  the 
trade  name  of  Pituitrin,  gives  the  Biuret-reaction  and  its  activity  is 
rapidly  destroyed  by  trypsin;  it  is  consequently  believed  to  be  a 
Peptamine  or  an  amine  derived  from  a  polypeptide  containing  a  histidine 
radical.  Several  synthetic  peptamines  have  been  prepared  but  their 
actions  have  hitherto  been  found  to  be  much  weaker  than  those  of  the 
simpler  amines. 

A  parent-material,  which  yields  pituitrin,  or  at  least  a  substance 
resembling  pituitrin  in  its  action  upon  the  uterus  after  hydrolysis  by 
acids  or  alkalies  (Fig.  6),  is  found  in  the  Anterior  Lobe  or  glandular 
portion  of  the  pituitary  body.  This  substance,  which  is  a  water- 
soluble  phospholipin,  has  been  designated  Tethelin.  The  histological 
structure  and  anatomical  relationship  of  the  two  parts  of  t  he  pituitary 
body  are  such  as  to  suggest  that  the  anterior  lobe  furnishes  some 
material  to  the  posterior  lobe,  and  it  is  therefore,  possible  that  the 
posterior  lobe  manufactures  pituitrin  from  tethelin  supplied  to  it  by 
the  anterior  lobe. 

A  nitrogenous  base  of  unknown  composition  appears  to  be  the  active 
principle  in  acidified  aqueous  extracts  of  intestinal  mucosa  which 
stimulates  the  secretion  of  Pancreatic  Juice  when  these  extracts  are 
injected  intravenously.  The  substance  which  is  known  as  Secretin, 
is  insoluble  in  neutral  water,  soluble  in  dilute  acids,  and  precipitable 
by  mercuric  chloride  and  by  picric  acid. 


REFERENCES. 
GENERAL: 

Jones:     The  Nucleic  Acids.     London,  1914. 
Barger:     The  Simpler  Natural  Bases.     London,  1914. 
THE  NUCLEIC  ACIDS: 

Jones  and  Rowntree:     Jour.  Biol.  Chem.,  1908,  4,  p.  289. 

Jones  and  Richards:     Ibid.,  1914,  17,  p.  71. 

Wells:    Ibid.,  1916-1917,  28,  p.  11. 

Jones  and  Read:     Ibid.,  1917,  29,  pp.  Ill  and  123;  1917,  31,  pp.  39  and  337. 

Read:     Ibid.,  1917,  31,  p.  47. 

Read  and  Tottingham:     Ibid.,  1917,  31,  p.  295. 

Levene:     Ibid.,  1917,  31,  p.  591. 

VlTAMINES: 

Funk:     Ergeb.  d.  Physiol.,  1913,  13,  p.  125. 

Folin  and  Macallum:     Jour.  Biol.  Chem.,  1912,  11,  p.  265. 

Funk  and  Macallum:     Biochem.  Jour.,  1913,  7,  p.  356. 

Williams:     Jour.  Biol.  Chem.,  1916,  25,  p.  437;  1917,  29,  p.  495. 

Williams  and  Seidell:     Ibid.,  1916,  26,  p.  431. 

Voegtlin  and  Myers:     U.  S.  Pub.  Health  Service.     Reprint  No.  471.     Pub.  Health 

Reports,  Washington,  1918. 
PHYSIOLOGICAL  ACTIONS  OF  THE  BASES. 

Sollmann:     A  Manual  of  Pharmacology.     Philadelphia,  1917. 

Cushny:     A  Text-book  of  Pharmacology  and  Therapeutics.     Philadelphia,  1918. 

Schafer:     The  Endocrine  Organs.     New  York,  1916. 

Maxwell:     Jour.  Biol.  Chem.,  1907,  3,  p.  359. 

Guggenheim:     Biochem.  Zeit.,  1913,  51,  p.  369;  1914,  65,  p.  189. 

Schmidt  and  May:     Jour.  Lab.  and  Clin.  Mod.,  1916-17,  2,  p.  708. 


CHAPTER  X. 
THE  HYDROLYZING  ENZYMES. 

GENERAL  CHARACTERISTICS  OF  THE  ENZYMES. 

Disaccharides  in  aqueous  solution  are  hydrolyzed  by  mineral  acids 
in  accordance  with  the  equation: 

Ci2H22On     +     H2O      =     2C6Hi2O6 

Any  acid  will  act  upon  any  disaccharide,  but  the  intensity  or  Velocity 
of  Hydrolysis  varies  somewhat  with  the  nature  of  the  acid  and  of  the 
disaccharide. 

The  law  which  connects  the  time  and  the  extent  of  the  hydrolysis 
of  cane-sugar  by  acids  was  first  formulated  by  Wilhelmy  in  1850,  who 
showed  that  at  every  instant  the  same  percentage  of  the  hitherto  unchanged 
sugar  is  hydrolyzed  per  second.  Thus,  if  to  begin  with  we  have  100 
parts  of  sugar,  and  of  this  5  parts  are  hydrolyzed  in  a  given  interval 
of  time,  we  have  now  95  parts  of  unchanged  sugar  left,  and  in  the 
succeeding  interval  five  hundred ths  of  this  will  be  hydrolyzed.  The 
transformation,  therefore,  proceeds  in  the  following  manner: 

Number  of  cane-  Number  of  sugar- 

sugar  molecules.  molecules  hydrolyzed. 

First  interval  of  time    .      .      .      .      .      100.00  T^7   X   100.00   =  5.00 

Second  interval  of  time      .   .  .      .      .  95.00  Y^  X  95.00  =  4.75 

Third  interval  of  time  .      .      .      .      .  90.25  Tfo  X  90.25  =  4.51 

Fourth  interval  of  time      ....  85.74  T^  X  85.74  =  4.29 

Fifth  interval  of  time   .....  81.45  Tfo  X  81.45  =  4.07 

In  other  words,  unit-mass  of  sugar,  or  one  hundred  molecules  of 
sugar,  always  decomposes  at  the  same  rate,  no  matter  how  much  or 
how  little  sugar,  i.e.,  how  many  units  or  what  fraction  of  a  unit,  is 
present  in  the  given  solution  at  the  moment.  If  a  gram-molecule 
of  sugar  be  present,  just  the  same  percentage  of  sugar-molecules  will  be 
undergoing  transformation  per  second  as  when  five  gram-molecules 
of  sugar  are  present,  but  in  the  latter  case  the  total  amount  of  trans- 
formation observed  per  second  will  be  five  times  as  great  as  in  the 
former. 


202  THE  HYDROLYZING  ENZYMES 

The  rationale  of  this  law,  which  is  known  as  Wilhelmy's  Law,  may 
be  made  clear  in  the  following  way:  In  every  instant  innumerable 
collisions  are  taking  place  between  sugar-molecules  and  water-molecules. 
Only  a  small  proportion  of  these  collisions  are  effective  in  accomplishing 
the  breaking  up  of  a  disaccharide  molecule.  The  proportion  of  effective 
collisions  is,  however,  the  same  no  matter  how  many  sugar-molecules 
may  chance  to  be  present.  We  may  picture  to  ourselves,  without 
seeking  to  employ  the  analogy  too  literally,  the  effective  collisions  as 
"head-on"  collisions,  the  ineffective  collisions  being  "glancing." 
Each  sugar-molecule  is  independent  of  all  the  rest,  and  its  chance  of 
achieving  an  effective  collision  with  a  water-molecule  is  the  same  as 
that  of  all  the  rest.  Suppose  every  thousandth  collision  is  effective, 
i.  e.,  one  tenth  of  a  per  cent,  of  the  total  collisions  per  second.  If  the 
solution  of  sugar  be  2-molecular,  the  total  number  of  collisions  per 
cubic  centimeter  per  second  will  be  twice  as  great  as  when  the  solution 
is  1 -molecular,  because  there  are  twice  as  many  molecules  of  sugar  in  a 
given  space.  In  each  solution  the  percentage  of  effective  collisions  is 
the  same.  Out  of  one  thousand  collisions  in  a  2-molecular  solution  one 
will  be  effective,  and  out  of  a  thousand  collisions  in  a  1-molecular  solu- 
tion one  will  also  be  effective.  But  as  there  are  twice  as  many  collisions 
per  second  in  the  former  as  there  are  in  the  latter  solution,  there  will 
also  be  twice  as  many  effective  collisions  per  second,  and  the  amount  of 
sugar  transformed  in  a  second,  i.  e.,  the  Velocity  of  Hydrolysis  in  the 
2-molecular  solution  must  be  twice  as  great  as  it  is  in  the  1-molecular 
solution. 

This  very  simple  relationship  may  also  be  expressed  in  an  algebraical 
formula : 

Velocity  of  hydrolysis      =     k(a — x) 

where  (a — x)  is  the  mass  of  unaltered  sugar  at  any  instant,  "a"  being 
the  initial  amount  and  "x"  the  quantity  which  has  already  undergone 
hydrolysis  at  the  moment  of  observation.  The  constant  "k"  expresses 
the  constant  ratio  which,  as  we  have  seen,  subsists  between  the  mass 
of  unhydrolyzed  sugar  which  is  present  and  the  velocity  with  which 
hydrolyzed  sugar  is  making  its  appearance.  It  is,  in  fact,  the  velocity 
of  hydrolysis  when  (a — x)=l,  that  is,  when  the  mass  of  unconverted 
sugar  is  unity,  one  gram-molecule,  or  one  gram,  or  whatever  mass 
we  may  arbitrarily  choose  as  a  unit,  provided  we  measure  all  the 
quantities  in  the  equation  in  the  terms  of  the  same  unit.  Also,  and 
this  is  very  important  to  notice,  the  constant  "k"  is  a  direct  measure 
of  the  percentage  of  effective  collisions  between  sugar-molecules  a  ad 
water-molecules,  for  if  the  percentage  of  effective  collisions  be  doubled 
by  any  means  then  the  velocity  of  hydrolysis  must  obviously  be 
doubled  also.  In  the  equation : 

Velocity     =     k(a — x) 


GENERAL  CHARACTERISTICS  OF  THE  ENZYMES  203 

if  the  term  "velocity"  is  doubled  in  magnitude,  while  (a — x)  remains 
unaltered,  then  k  must  have  been  doubled  in  magnitude.  When  the 
percentage  of  effective  collisions  is  doubled,  therefore,  k  is  doubled, 
and  so  forth. 

We  have  hitherto  not  considered  the  part  which  the  Acid  plays  in 
bringing  ahout  the  hydrolysis.  Neutral  water  only  hydrolyses  sugar 
extremely  slowly,  so  slowly  that  the  velocity  is  negligible  in  comparison 
with  the  velocity  of  hydrolysis  in  acid  solutions.  At  the  completion  of 
hydrolysis  the  acid  is  unaltered  and  is  available  for  bringing  about 
further  and,  apparently,  unlimited  hydrolysis.  The  acid  does  not, 
therefore,  communicate  any  energy  to  the  system;  it  merely  increases 
the  Percentage  of  Effective  Collisions  of  molecules  of  sugar  with  mole- 
cules of  water.  Such  an  action  is  termed  a  Catalytic  Action,  and  the 
agent  which  brings  about  the  acceleration,  in  this  instance  the  acid, 
is  termed  a  Catalyzer  or  Catalyst.  The  Mechanism  of  Catalysis  is,  in 
this  instance,  not  perfectly  clear,  but  judging  from  the  analogy  afforded 
by  the  mode  of  action  of  many  other  catalysts,  we  may  conclude  that 
a  compound  of  the  disaccharide  with  acid  is  formed  and  that  it  is  this 
compound  which  actually  undergoes  hydrolysis.  In  many  cases  of 
catalysis  such  compounds  of  the  catalyst  with  the  substance  undergoing 
decomposition,  or  Substrate,  have  been  isolated  and  identified,  so  that 
we  feel  justified  in  assuming  that  if  such  compounds  are  not  readily 
detectable  in  an  instance  of  catalysis  such  as  that  afforded  by  the 
hydrolysis  of  cane-sugar  by  acids,  the  reason  is  that  only  a  minute 
trace  of  the  compound  of  the  catalyst  and  the  substrate  is  present  in 
the  mixture  at  any  moment 

The  quantity  of  the  compound  of  the  substrate  with  the  catalyst 
which  is  present  at  any  moment  in  the  mixture,  must,  however  small, 
be  proportional  to  the  concentration  of  the  catalyzer  and  also  to  the 
concentration  of  the  substrate,  for  if  two  substances  A  and  B  combine 
to  form  a  third  AB  the  quantity  of  this  compound  formed  must  be 
determined,  as  the  Guldberg  and  Waage  Mass-law  requires,  by  the 
equation : 

Mass  of  A  X  mass  of  B      =     constant  X  mass  of  AB 

If,  then,  the  concentration  (=mass  per  unit- volume)  of  the  catalyzer 
be  kept  constant,  the  quantity  of  the  substrate-catalyzer  compound, 
in  this  instance  the  compound  of  cane-sugar  with  acid,  must  be  directly 
proportional  to  the  concentration  of  the  still  unaltered  cane-sugar  and 
decrease  as  it  decreases.  Since,  for  all  practical  purposes  of  measure- 
ment, it  is  only  the  molecules  of  sugar-acid  compound  which  are  under- 
going hydrolysis  it  follows  that  the  velocity  of  hydrolysis  must,  if  the 
same  proportion  of  these  compound  molecules  is  decomposed  in  each 
instant,  fall  off  in  direct  proportion  to  the  concentration  of  still  un- 
altered sugar,  or  in  other  words  the  equation : 

Velocity  of  hydrolysis      =     k(a    —  x) 


204  THE  HYDROLYZING  ENZYMES 

must  hold  good  for  a  catalyzed  as  for  an  uncatalyzed  hydrolysis,  so 
long  as  the  concentration  of  the  catalyzer  is  kept  unaltered. 

Since  the  quantity  of  the  acid-sugar  compound  is  also  proportionate 
to  the  concentration  of  the  acid,  it  follows  that  with  varying  concen- 
trations of  acid  the  velocity  of  hydrolysis  must  also  vary  directly 
with  the  concentration  of  acid.  In  other  words  the  value  of  "k"  in 
the  above  equation  is  directly  proportional  to  the  concentration  of 
catalyst,  or: 

Velocity  of  hydrolysis      =     kf  (a    —  x) 

where  "f"  is  the  concentration  of  the  catalyzer.  In  fact  so  accurately 
does  this  relationship  obtain  that  the  ratio  of  the  velocity  of  the 
hydrolysis  of  cane-sugar  to  its  concentration  is  very  commonly 
employed  as  a  means  of  measuring  the  quantity  of  free  acid  (= hydrogen 
ions)  which  is  present  in  a  solution. 

So  much  for  the  hydrolysis  of  cane-sugar  and  of  other  disaccharides 
by  acids;  but  cane-sugar  is  also  hydrolyzed  by  an  Enzyme,  to  wit, 
Invertase.  This  enzyme  is  found  in  yeasts,  in  certain  moulds  and 
bacteria,  in  green  leaves  and  young  twigs,  in  some  fruits  and  in  germi- 
nating grains.  In  mammalia,  it  is  sometimes  found  in  the  human 
gastric  juice,  but  not  in  the  gastric  juice  of  cows.  It  is  also  found  in 
weak  concentration  in  other  organs.  It  may  be  extracted  from  yeast- 
cells  with  water,  provided  the  cells  have  previously  been  subjected  to 
the  action  of  some  Plasmolyzing  Agent,  or  agent  which  will  break  up  or 
enhance  the  permeability  of  the  limiting  membrane  of  the  cell,  such  as 
alcohol  or  ether.  The  invertase  can  then  be  precipitated  from  its 
watery  solution  by  alcohol,  and  this  precipitate,  in  nearly  neutral 
solutions  in  which  hydrolysis  would  otherwise  be  excessively  slow, 
rapidly  decomposes  cane-sugar  into  its  constituent  hexoses. 

It  should  be  clearly  understood  that,  as  in  the  case  of  the  other 
enzymes,  we  possess  no  clue  which  enables  us  to  decide  whether  there 
is  only  one  or  whether  there  are  many  invertases.  We  do  not  know 
anything  whatever  concerning  the  chemical  composition  of  invertase. 
Our  only  means  of  recognizing  this  enzyme  is  by  the  property  which  it 
possesses  of  hydrolyzing  cane-sugar,  giving  rise  to  Glucose  and  Fructose. 
Any  agent  which  can  be  extracted  from  living  tissues  which  does  this 
and  is  inactivated  by  high  temperatures  we  call  invertase.  It  is  quite 
conceivable  that  many  different  substances  can  accomplish  this 
hydrolysis.  The  activity  of  an  invertase  preparation  is  no  guide  to  its 
individuality,  because  in  the  absence  of  any  knowledge  of  the  chemical 
properties  of  invertase,  we  cannot  estimate  the  purity  of  any  prepara- 
tion. We  can  recognize  certain  impurities,  such  as  phosphates,  pro- 
teins and  so  forth  in  a  preparation  of  invertase,  and  we  can  remove 
some  of  them,  wholly  or  partially.  But  when  easily  recognizable 
impurities  have  been  removed,  we  cannot  tell  whether  the  residuum  is  a 
pure  material,  that  is,  a  chemical  individual,  or  a  mixture  of  different 


GENERAL  CHARACTERISTICS  OF  THE  ENZYMES          205 

substances  of  which  perhaps  one  is  active  or,  perhaps,  many.  Could 
we  discover  any  chemical  resembling  invertase  in  its  solubilities  and 
in  its  sensitiveness  to  temperature  and  so  forth,  and  possessing  the 
action  of  invertase,  we  might  be  inclined  to  claim  an  identity  between 
the  two  and  then  analysis  would  give  us  a  criterion  of  the  purity  of  any 
given  invertase  preparation.  But  at  present  we  have  no  such  criterion, 
and  we  cannot  say  whether  a  given  preparation  of  invertase  is  a  single 
or  a  multiple  preparation,  or  whether  it  contains  99  per  cent,  of  inver- 
tase and  1  per  cent,  of  impurities  or,  on  the  contrary,  1  per  cent,  of 
invertase  and  99  per  cent,  of  impurities. 

In  this  connection  the  properties  and  peculiarities  of  invertase  may 
be  regarded  as  illustrative  of  the  peculiarities  which  distinguish  nearly 
all  of  the  enzymes.  The  same  difficulties  are  encountered  in  the  study 
of  each  of  the  enzymes  in  turn.  It  might  be  imagined  that  the  problem 
of  extracting  an  enzyme  in  pure  condition  from  a  crude  preparation  of 
proved  activity  could  be  attacked  in  a  manner  analogous  to  that 
employed  in  the  original  discovery  of  radium;  by  employing  a  variety 
of  precipitant s  and  solvents  to  fractionate  the  crude  preparation,  reject- 
ing the  inactive  and  retaining  the  active  fraction  in  each  successive 
stage  of  the  process.  Unfortunately,  however,  it  is  found  that  almost 
every  chemical  procedure  which  we  may  employ  results  in  some  loss 
of  activity  by  the  enzyme.  If  a  fraction  of  the  original  crude  pre- 
paration be  precipitated  out  from  the  rest  it  may  be  found,  for  example, 
to  contain  an  amount  of  active  ferment  corresponding  to  fifty  per  cent, 
of  the  amount  which  was  present  in  the  crude  preparation,  while  the 
residue,  after  the  separation  of  the  precipitate,  may  be  found  to  contain 
none  of  the  ferment  whatever.  In  this  way  successive  processes  of  puri- 
fication involve  successive  losses,  until  the  activity  of  the  preparation 
ultimately  disappears  altogether.  It  is  for  this  reason  that  the  more 
impure  preparations  of  the  various  soluble  enzymes  are  usually  more 
active  than  those  preparations  which  are  relatively  "pure,"  i.  e.,  contain 
a  smaller  variety  of  substances. 

We  have  referred  to  the  fact  that  many  different  enzymes  may  be 
mistakenly  regarded  as  one  if  they  chance  to  possess  a  common  action. 
As  an  illustration  of  how  an  enzyme  regarded  as  a  single  chemical 
individual  may  become,  with  increased  acquaintance,  recognized  as 
multiple,  we  may  cite  the  Trypsins :  enzymes  which  have  this  in  com- 
mon— that  they  hydrolyze  proteins  and  peptones  in  faintly  alkaline 
solutions.  Until  recently  no  means  of  distinguishing  between  dif- 
ferent trypsins  were  known,  and  trypsin  was  tacitly  assumed  to  be 
one  ferment  and  only  one.  Now  the  investigations  of  Emil  Fischer 
and  of  Abderhalden  have  shown  us  that  there  are  many  trypsins,  which 
differ  from  one  another  in, the  relative  ease  with  which  they  attack 
different  peptide-linkages. 

The  enzymes  are,  as  a  rule,  destroyed,  or,  at  least,  inactivated  by 
high  temperatures.  But  this  is  by  no  means  a  rule  without  exceptions. 


206  THE  HYDROLYZING  ENZYMES 

Thus  several  of  the  oxidizing  enzymes,  or  Oxidases  regain  their  activity, 
lost  by  heating,  when  the  solution  is  allowed  to  stand  for  some  time  at 
ordinary  temperatures.  According  to  Gramenetski  the  same  phe- 
nomenon may  be  displayed  by  certain  Diastases  or  starch-splitting 
enzymes  and  even  in  some  measure  by  Trypsin.  The  vegetable 
Proteases  or  protein-splitting  enzymes  sometimes  withstand  higher 
temperatures  than  the  corresponding  enzymes  of  animal  origin  and 
Karl  Meyer  has  drawn  attention  to  the  rather  extraordinary  fact  that 
the  Trypsin  which  is  produced  in  culture-media  by  Bacillus  prodigiosus 
will  withstand  heating  for  fifteen  minutes  to  100°  C,  although  it  is 
destroyed  within  thirty  minutes  at  56°  C.  This  looks  rather  as  though 
a  trypsin-splitting  ferment  also  existed  in  the  culture-medium  for 
which  the  optimum  temperature  is  about  56°,  and  which  is  destroyed 
by  higher  temperatures  more  rapidly  than  trypsin  itself. 

It  would,  therefore,  be  very  unsafe  to  infer  because  a  substance  does 
not  lose  its  characteristic  activity  of  some  type  or  other  when  it  is 
heated  that  it  is  therefore  not  an  enzyme.  It  would  be  still  more 
unsafe,  of  course,  to  infer  that  it  is  an  enzyme  simply  because  it  is 
"inactivated"  by  heat.  Yet  both  of  these  inferences,  unfortunately, 
have  not  infrequently  been  made  in  biological  and  biochemical  investi- 
gations. In  deciding  whether  or  not  a  substance  or  material  should 
be  classed  as  an  enzyme  we  should  be  guided,  rather,  by  its  quantitative 
relationship  toward  the  particular  activity  which  it  displays.  The 
enzymes  are  usually  effective  in  relatively  minute  concentration.  It 
has  been  estimated,  for  example,  that  a  certain  Rennin  or  milk-coagulat- 
ing enzyme  preparation  will  convert  no  less  than  500,000  times  its 
weight  of  casein  into  the  coagulating  form,  paracasein,  and  a  prepara- 
tion of  pepsin  has  been  obtained  which  will  hydrolyze  to  peptones 
100,000  times  its  weight  of  fibrin.  The  excessive  amount  of  change 
which  may  thus  be  brought  about  by  relatively  minute  proportions  of 
enzymes  almost  compels  the  assumption  that  they  are  not  consumed 
during  the  progress  of  the  changes  which  they  accelerate,  for  otherwise 
the  enzyme  would  probably  be  "used  up"  long  before  so  immense  a 
proportion  of  change  had  been  accomplished.  It  is,  however,  impos- 
sible at  the  present  stage  of  our  knowledge  to  submit  this  supposition 
to  vigorous  investigation,  because  the  various  hydrolyzing  enzymes, 
at  all  events,  are  themselves  chemically  unstable  substances  and 
undergo  spontaneous  transformation  resulting  in  inactivation  on 
standing  in  aqueous  solution.  They  are  carried  down  together  with 
any  precipitates  which  may  be  formed  in  the  digestion-mixture  and 
are  not  infrequently  partially  bound  by  or  combined  with  not  only  the 
substrate,  but  also  the  products  of  hydrolysis.  Any  chemical  procedure 
designed  to  isolate  and  recover  the  enzyme  from  a  digest  in  which  it  has 
been  operating  would  involve  loss  or  impairment  of  the  enzyme  even 
if  it  had  been  dissolved  in  distilled  water  instead  of  in  a  solution  of  the 
substrate  which  it  attacks  and  of  the  products  of  its  hydrolysis.  No 


QUANTITATIVE  RELATIONSHIPS  IN  HYDROLYSIS         207 

attempt  to  re-isolate  an  enzyme  after  it  has  acted  upon  a  measured 
amount  of  substrate,  in  order  to  determine  the  loss  of  activity  it  may 
or  may  not  have  sustained  during  the  reaction,  can  possibly  be  success- 
ful, therefore,  in  the  present  inadequate  state  of  our  knowledge  and  our 
manipulative  technique. 

A  variety  of  the  hydrolyzing  enzymes  are  not  only  inactivated, 
temporarily  or  permanently  by  heat,  but  also  by  exposure  to  light 
and  particularly  to  Ultraviolet  Light.  Solutions  of  enzymes  are  also 
temporarily  inactivated  by  intense  agitation  of  the  solution  of  such  a 
character  as  to  give  rise  to  excessive  formation  of  foam.  It  is  then 
found  that  the  enzyme  has  become  concentrated  in  the  foam  and  is 
restored  to  the  solution  when  the  foam  subsides.  A  portion  of  the 
enzyme  also,  under  these  circumstances,  becomes  temporarily  attached 
to  the  surface  of  the  containing  vessel.  This  phenomenon  is  not 
peculiar  to  enzymes,  however,  for  it  is  exhibited,  in  greater  or  less 
measure,  by  all  those  substances  which,  when  dissolved  in  water,  reduce 
the  Surface-tension  of  an  air-water  interface.  For  example  it  is  dis- 
played to  a  striking  extent  by  the  various  Saponins  or  by  Bile-salts. 
(See  Chapter  XIII.)  This  liability  to  become  concentrated  at  liquid 
surfaces  is  probably  the  explanation  of  the  striking  tendency,  to  which 
reference  has  already  been  made,  of  the  various  hydrolyzing  enzymes 
to  be  carried  down  in  association  with  precipitates  which  form  in  their 
solutions.  They  are  similarly  "adsorbed"  by  such  substances  as 
animal  charcoal  or  by  insoluble  proteins. 


THE  QUANTITATIVE  RELATIONSHIPS  IN  HYDROLYSIS  BY 

ENZYMES. 

We  have  seen  that  when  cane-sugar  is  hydrolyzed  by  acids  the 
relationship  between  the  amount  of  unaltered  sugar  in  the  system  and 
the  velocity  of  change  is  rather  a  simple  one.  The  two  quantities,  the 
amount  of  unaltered  sugar  and  the  velocity  of  decomposition,  are 
simply  proportional  to  one  another  and  stand  in  a  constant  ratio  to 
one  another  throughout  the  reaction.  The  case  is  not  so  simple  when 
the  hydrolysis  is  brought  about  by  Invertase.  It  will  be  recollected 
that  we  regard  the  rapid  hydrolysis  of  cane-sugar  by  acids  as  being  due 
to  the  formation  of  a  compound  between  the  cane-sugar  and  the  acid, 
this  compound  being  very  easily  attacked  by  water.  At  any  instant 
the  percentage  of  acid  thus  combined  is  almost  infinitesimal.  The 
amount  of  this  compound  is,  as  usual  in  such  cases,  directly  propor- 
tional to  the  concentrations  of  its  components,  the  acid  and  the  sugar, 
so  that  we  have: 

Concentration  of  sugar-acid  compound  =  constant   X  concentration  of  acid 
X  concentration  of  sugar. 


208  THE  HYDROLYZING  ENZYMES 

The  velocity  of  hydrolysis  is  proportional,  at  every  instant,  to  the 
concentration  of  that  portion  of  the  sugar  which  is  actually  undergoing 
hydrolysis,  i.  e.,  the  sugar-acid  compound,  and  so  we  have: 

Velocity  of  hydrolysis  =  constant  X  concentration  of  sugar-acid  compound. 

Combining  the  two  equations  we  find  that : 

Velocity  of  hydrolysis  =  k  X  concentration  of  acid  X  concentration  of  sugar. 

For  any  given  concentration  of  acid,  therefore,  since  the  acid  is  not 
consumed  at  all  during  the  reaction,  we  have : 

Velocity  of  hydrolysis     =     k(a   —  x) 

where  (a  —  x)  is  the  concentration  of  unhydrolyzed  sugar  at  any  instant. 

The  case  would  be  very  different,  however,  if  more  than  a  trace  of 
the  catalyst  were  combined  with  the  sugar.  Suppose,  for  example,  that 
in  the  early  stages  of  the  reaction,  all  of  the  catalyst  were  combined 
with  the  sugar;  then,  so  long  as  there  were  enough  sugar  present  to 
combine  with  all  of  the  catalyst  the  amount  of  the  catalyst-sugar 
compound  would  always  be  the  same,  namely  the  chemical  equivalent 
of  the  amount  of  the  catalyst  in  the  mixture.  But  since  this  is  the 
only  portion  of  the  sugar  which  hydrolyzes  at  any  measurable  rate,  the 
velocity  of  hydrolysis  would  in  that  event  be  constant. 

This  will,  of  course,  only  hold  good  so  long  as  the  sugar  which  is 
still  unconverted  is  sufficient  to  combine  with  all  of  the  catalyzer. 
As  the  reaction  proceeds,  however,  a  point  will  be  reached  at  which 
the  amount  of  sugar  is  insufficient  to  bind  all  of  the  catalyzer.  After 
this  point  in  the  reaction  is  reached,  the  amount  of  the  catalyst  bound 
by  the  sugar,  and  therefore  the  velocity  of  hydrolysis  of  the  sugar,  will 
become  progressively  less  as  the  conversion  proceeds.  In  fact  it  will 
obviously  be  equal  to  the  quantity  of  sugar  which  is  still  unconverted, 
and  again  we  shall  have  the  relationship: 

Velocity  of  hydrolysis  =  k  X  concentration  of  unhydrolyzed  sugar 
for  any  given  proportion  of  acid  present  in  the  mixture. 

These  complications,  as  has  been  implied  above,  are  not  observed 
during  the  hydrolysis  of  cane-sugar  by  acids  because  the  proportion  of 
acid  which  is  at  any  instant  bound  by  the  sugar  is  so  small  that  it  has 
not  as  yet  been  quantitatively  estimated.  But  when  Invertase  is  the 
catalyst  instead 'of  acid,  we  meet  with  precisely  the  conditions  which 
we  have  outlined.  When  the  proportion  of  sugar  to  invertase  is  high, 
all  of  the  invertase  is  bound  by  the  sugar,  and  the  portion  of  the  sugar 
which  is  thus  combined  is  the  only  portion  which  undergoes  hydrolysis 
at  a  perceptible  rate.  Under  such  conditions,  for  a  given  concentration 
of  invertase,  the  rate  of  hydrolysis  of  the  sugar  is  constant,  while  for 
varying  amounts  of  invertase,  the  velocity  of  hydrolysis  is  proportional 
to  the  concentration  of  invertase  employed.  Algebraically: 

Velocity     =     kF 


QUANTITATIVE  RELATIONSHIPS  IN  HYDROLYSIS         209 

where  "k"  is  a  constant  and  "F"  is  the  concentration  of  the  invertase. 
As  the  proportion  of  sugar  to  enzyme  falls  off,  however,  a  point  must 
be  reached  at  which  the  remnant  of  sugar  is  insufficient  to  bind  all  of 
the  invertase.  At  this  stage  of  the  reaction,  therefore,  or  if  the  pro- 
portion of  sugar  to  enzyme  is  small  to  begin  with,  the  reaction-velocity 
begins  to  fall  off  as  the  reaction  proceeds,  in  accordance  with  the 
formula  : 

Velocity  =     kF(a    —  x) 

the  numerical  value  of  "k"  being  different  in  the  two  cases  because, 
in  the  latter  case,  the  "k"  includes  the  equilibrium  constant  for  the 
reaction  : 

Sugar     +     catalyst     *~j     sugar-catalyst  compound. 

Turning  now  to  the  question  of  the  relationship  of  the  Quantity  of 
Substrate  hydrolyzed  to  the  Time  of  Hydrolysis  it  is  at  once  manifest 
that  if  the  relationship 

Velocity     =     kF 

holds  good,  then,  the  quantity  of  sugar  decomposed  in  each  unit  of 
time  being  the  same,  the  quantity  "x"  decomposed  after  time  "t" 
must  be  given  by  the  equation  : 

x    =    kFt 

In  the  more  general  case  in  which  the  relationship  obtains: 

Velocity      =     kF(a    -  x) 

the  relationship  between  the  mass  of  substrate  transformed  and  the 
time  occupied  in  the  transformation  may  be  found  by  a  simple  opera- 
tion of  the  integral  calculus  to  be  : 

loge  -^-     =     kFt 


The  following  results  are  illustrative  of  these  two  types  of  relation- 
ship: 
Invertase  on  cane-sugar  (A.  J.  Brown)  Concentrated  Substrate: 

Grams  of  cane 

sugar  per  Grams  of  cane-sugar 

100  c.c.  inverted  in  60  minutes. 

40.02  .      .      .     .    :.      .      .      .V.      .  1.076  0.179 

29.96  ...........  1.235  0.206 

19.91   ...........  1.355  0.226 

9.85   ...........  1.355  0.226 

4.89  ...........  1.230  0.206 

Invertase  on  cane-sugar  (A.  J.  Brown)  Dilute  Substrate: 

j 

Grams  of  cane-  10s            a 
sugar  per                                                                Grams  of  cane-sugar            1(PK  =  --  logio 

100  c  c.                                                                    inverted  in  60  minutes.  t 

(2.00)    ...........    (0.308)  (132) 

1.00     ...........      0.249  219 

0.50     ...........      0.129  239 

0.25     ...........      0.060  228 

14 


210  THE  HYDROLYZING  ENZYMES 

Saliva-Diastase  on  Starch  (A.  E.  Taylor) ;  a  =  0.25  per  cent.  Starch: 

10«K  = logic 


Time  in  minutes. 


t      a— x 


30  .   .   .  ';   .   .   .   .'*   •   .   .-  .  .   ,   •   •  490 

45 465 

60 455 

75 470 

90 •   •  465 

120 .455 

150 ;   •   v  460 

180  .   .   :   .   .....   .   •   •   •   •   •   •   •   •  ,  455 

Trypsin  on  d-alanyl-d-alanine  (Abderhalden  and  Koelker) : 

103X 

K  =  - 

Time  in  minutes.  t 

50                                       •      •'  •  v  380 

65  .400 

7.5 .  400 

16.0 369 

22.0 368 

28.0 368 

30.0 ,-.•-.'•  314 

38.0 ;  332 

Trypsin  on  Sodium  Caseinate  (E.  H.  Walters) : 

10*  a 

104K  = logio  — 

Time  in  minutes.  t  a  —  x 

15 18. 

30 16. 

45 : 15. 

60 -....,,....,  14. 

75 13. 

90       .....      ^     ...........  12.5 

105 12. 

120 .  12. 

135 12. 

150 13. 

165 .13. 

180 12.5 

210       .................  13. 

240 •'..'-• 12.5 

270       .      .    \     ;.....      .-..:.-    Y  ...     ...     ...  12. 

300  ..      .      .      .      .      .      .      .      .      .      .      .      .    :.      .      .      .  13.5 

330 ' .      .  13. 

360       .      .      .      .      .      ...      .      .      .      .      .      ...      .      .14. 

420 •  .    \      .'•.-.     V     .      .      .  14.5 

480 ;'•  .      .      .      .      .    ".      .     '.  " '..      .  14. 

540 .     .     ...     ....     .      .      .  14. 

In  certain  instances  yet  another  type  of  relationship  between  the 
amount  of  transformation  and  the  time  may  be  observed.  In  the 
clotting  of  milk  by  Rennin  and  in  the  hydrolysis  of  proteins  by  Pepsin 
we  find  that  the  mass  "x"  of  the  substance  transformed  is  connected 
with  the  time  "t,"  the  initial  concentration  of  the  substrate  "a,"  and 
the  concentration  of  the  enzyme  "F"  by  the  relationship: 


k\Art 


QUANTITATIVE  RELATIONSHIPS  IN  HYDROLYSIS         211 

which  is  known  as  the  Schiitz-Borissoff  Rule.  The  rule  only  holds  good, 
however,  during  the  earlier  stages  of  the  hydrolysis  and  before  about 
fifty  per  cent,  of  the  substrate  has  been  hydrolyzed.  Arrhenius  has 
found  that  an  exactly  similar  relationship  obtains  between  the  time 
and  the  extent  of  the  transformation  in  the  hydrolysis  of  Ethyl  Acetate 
by  ammonia.  He  accounts  for  it  by  the  fact  that  in  this  instance,  and 
presumably  also  in  pepsin-digests,  the  catalyzer  is  bound  and  inactiv- 
ated by  one  of  the  products  of  the  hydrolysis;  thus  ammonia  combines 
with  the  acetic  acid  which  is  liberated  by  the  hydrolysis  of  ethyl  acetate, 
forming  ammonium  acetate  which  exerts  no  catalytic  action. 

If  we  differentiate  the  above  algebraical  expression  of  the  Schiitz- 
Borissoff  rule  we  obtain: 

dx  k2     aF 

—      =     velocity  of  hydrolysis     =     —     — 
dt  2       x 

from  which  it  is  evident  that  the  velocity  of  hydrolysis  is  inversely 
proportional  to  its  extent.  We  can  account  for  this  by  supposing  that 
the  enzyme  combines  with  a  product  of  the  hydrolysis  to  form  an 
inactive  compound  according  to  the  equation: 

Free  enzyme      +     product      =     inactive  compound 

and  that  the  concentration  of  enzyme  is  so  small  compared  with  that  of 
the  substrate  that  in  the  first  few  moments  after  digestion  has  begun 
the  concentration  of  the  inactive  compound  is  nearly  equal  to  the 
whole  of  the  initial  concentration  of  the  enzyme.1  The  trace  of  free 
and  active  enzyme  which  then  remains  will  be  given  by: 

initial  concentration  of  enzyme 

Concentration  of  free  enzyme    =  constant    X  -  — 7: 7 1 — 

concentration  of  product 

or,  expressed  algebraically: 

kF 

Concentration  of  free  enzyme    =  — 


The  actual  velocity  of  hydrolysis  must  be  proportional  to  the  con- 
centration of  active  catalyst  and  also  to  the  concentration  of  uncon- 
sumed  substrate;  hence  we  have: 

kF 
Velocity  of  hydrolysis    =   --  k*(a   —  x) 

where  "k1"  is  a  proportionality-factor  which  differs  in  meaning  and 
magnitude  from  "k." 

1  These  conditions  obviously  do  not  hold  in  a  mixture  of  ethyl  acetate  and  ammonia, 
but  the  progressive  modification  of  the  electrolytic  dissociation  of  the  residual  ammonia 
by  the  ammonium  acetate  which  is  formed  during  the  reaction  brings  about  very  similar 
quantitative  relationships.  The  conditions  depicted,  however,  correspond  much  more 
closely  to  those  which  we  may  reasonably  expect  to  obtain  in  a  mixture  such  as  that 
furnished  by  a  pepsin-digest  than  to  those  which  actually  obtain  in  a  mixture  of  ammonia 
and  ethyl  acetate. 


212  THE  HYDROLYZING  ENZYMES 

The  integration  of  this  equation  would  lead  to  the  relationship 

loge  -        -   -  x    =  kFt 


which  can  be  shown  in  many  instances  to  be  the  relationship  which 
actually  does  obtain.  When  x  is  small,  however,  that  is,  in  the  early 
stages  of  the  digestion,  "a — x"  is  nearly  equal  to  "a;"  the  velocity 
equation  becomes: 

kFa 
Velocity  of  hydrolysis    =    - 

and  the  integrated  expression  becomes: 


x      =     \/2kFat 

which  is  the  Schiitz-Borissoff  rule. 

The  following  is  an  instance  of  the  applicability  of  these  relationships: 

DIGESTION  OF  EGG-ALBUMIN  BY  PEPSIN  FOLLOWED  BY  THE  CHANGES 
IN  THE  ELECTRICAL  CONDUCTIVITY  OF  THE  MIXTURE  (J.  SJOQVIST). 

log  — x 


Protein  ka   =    —  —    ks    = — 

Hours.  digested  /y/t 

2  .      .     ,     ~      .      .      .  10.5  3.0  7.5 

4  .      .      .     '.      .      .-•:,.  16.4  3.8  8.2 

6 19.9  3.8  8.1 

8  .      .      .      .      .      .      .  22.7  3.8  8.0 

12 27.0  3.7  7.7 

16  .......  30.4  3.6  7.6 

20  ...'....  33.7  3.7  7.5 

32  .      .      .      .      .      .      .  40.0  3.4  7.1 

48  ........  45.1  3.2  6.5 

64  ...      .      .   .-.      .  50.8  3.1  6.3 

96  .......  57.4  2.8  5.9 

Both  formulae  give  tolerably  uniform  values  for  the  constants,  but 
those  obtained  by  the  complete  logarithmic  formula  are  more  nearly 
uniform  than  those  obtained  by  the  employment  of  the  partial  expres- 
sion, the  Schiitz-Borissoff  rule. 

Whatever  may  be  the  relationship  between  the  extent  of  transforma- 
tion and  the  time  which  may  chance  to  obtain  in  a  given  instance  of  an 
enzymatic  hydrolysis,  one  quantitative  relationship  remains  invariably 
true,  namely,  that  the  time  required  to  attain  a  given  amount  of  trans- 
formation of  the  substrate  is  inversely  proportional  to  the  concentration  of 
the  enzyme.  There  appears  to  be  no  deviation  from  this  rule  which  is 
not  immediately  explicable  by  decomposition  of  the  enzyme  or  such 
adventitious  factors  as  fluctuation  of  temperature,  reaction  and  so 
forth.  The  following  are  instances  of  the  applicability  of  this,  the 
only  universal  rule  which  has  been  found  to  govern  the  action  of  the 
hydrolyzing  enzymes :  j 


INFLUENCE  OF  TEMPERATURE  UPON  ENZYMES        213 

TIME  REQUIRED  TO  LIQUEFY  GELATIN  HARDENED  BY  THYMOL,  WITH 
VARYING  QUANTITIES  OF  TRYPSIN  (MADSEN  AND  WALBUM.) 

F  =  Concentration  t  =Time  required  ' 

of  Trypsin.  in  hours.                                                          Ft. 

0.105  0.5  0.052 

0.050  1.0  0.050 

0.027  2.0  0.054 

0.020  3.0  0.060 

0.015  4.0  0.060 

0.011  5.0  0.055 

0.009  6.0  0.054 

0.0072  8.0  0.058 

0.0060  10.0  0.060 

0.0037  16.0  0.059 

0.0032  18.0  O.C58 

0.0027  20.0  0.054 

0.0025  22.0  0.055 

0.0022  24.0  0.053 

COAGULATION  OF  MILK  BY  RENNET  (MADSEN  AND  WALBUM). 

F  =  Concentration  t  =Time  in 

of  Rennet.  minutes.                                                          Ft. 

8.00  4  32 

5.00  6  30 

3.30  9                                                         30 

1.90  12  23 

1.30  20  26 

0.70  30  21 

0.70  35  25 

0.50  50  25 

0.40  70  28 

0.32  80  26 

0.28  100  28 

0.25  120  30 

0.185  180  33 

0.167  •    200  40 


THE  INFLUENCE  OF  TEMPERATURE  UPON  ENZYMES. 

The  general  effect  of  increasing  temperature  upon  the  hydrolyzing 
enzymes  is  to  accelerate  their  action.  This  favorable  influence  is, 
however,  limited  by  the  fact  that  after  the  temperature  exceeds  a 
certain  optimum  the  auto-inactivation  of  the  enzyme,  which  always 
takes  place  at  a  perceptible  rate  in  enzyme-solutions,  even  at  ordinary 
temperatures,  becomes  so  rapid  as  to  more  than  counterbalance  the 
acceleration  of  its  hydrolyzing  action.  The  Optimum  Temperature  for 
enzyme  action  was  formerly  supposed  to  be  a  characteristic  of  each 
enzyme,  distinguishing  it  more  or  less  sharply  from  other  enzymes. 
We  now  recognize,  however,  that  while  in  some  measure  the  optimum 
temperature  does  characterize  certain  groups  of  enzymes,  yet  it  is 
greatly  influenced  by  a  variety  of  factors  other  than  the  nature  of  the 
enzyme  itself,  such  as  the  reaction  (acidity  or  alkalinity)  of  the  medium 
in  which  the  enzyme  is  dissolved;  the  concentration  of  the  enzyme  itself 
and  the  nature  and  concentration  of  the  substrate  upon  which  it  is 
acting.  It  is,  of  course,  impossible  to  render  all  of  these  conditions 


214  THE  HYDROLYZING  ENZYMES 

comparable  in  experiments  with  different  enzymes,  and  we  are  there- 
fore left  frequently  in  uncertainty  whether  the  observed  differences  in 
temperature-optimum  are  in  reality  due  to  specific  differences  between 
the  enzymes  investigated  or  are  not  simply  attributable  to  the  circum- 
stances attending  their  action.  In  the  very  great  majority  of  cases, 
however,  it  is  found  that  the  temperature-optimum  from  hydrolyzing 
enzymes  lies  very  slightly  above  the  body-temperature  of  the  warm- 
blooded animals,  namely  between  40°  and  45°  C.,  the  normal  tempera- 
ture of  man  being  37.8°  C.  and  the  temperature  of  birds  about  41°  C. 

This  remarkable  correspondence  is  certainly  not  accidental,  and  we 
may  infer  that  the  processes  which  have  brought  about  the  evolution 
of  the  warm-blooded  animals  from  cold-blooded  ancestors  has  consisted 
essentially  in  an  improvement  of  the  adaptation  of  the  more  recent 
forms  to  the  properties  of  their  enzymes,  whereby  swifter  transforma- 
tions and  exchanges  of  material  are  rendered  possible  without  at  the 
same  time  incurring  the  wasteful  expenditure  of  catalysts  which  would 
be  involved  by  still  higher  bodily  temperatures.  The  factor  which 
determines  the  bodily  temperature  of  the  warm-blooded  animals  is 
certainly  not  the  coagulation-temperature  of  their  tissue-proteins,  for 
that  lies  very  considerably  above  the  maximum  body-temperature 
which  is  observed  in  any  species.  The  cold-blooded  animals  and 
plants,  therefore,  are  handicapped  by  a  disharmony  between  the 
properties  of  their  enzymes  and  the  temperature  of  their  tissues. 
Whether  or  not  this  is  in  some  instances  compensated  for  by  greater 
specific  activity  of  their  enzymes  or  by  the  production  of  enzymes  in 
greater  quantity  is  a  question  which  the  data  at  present  in  our  posses- 
sion do  not  enable  us  to  answer. 

In  a  few  exceptional  cases  the  temperature-optimum  lies  far  above 
the  usual  level.  We  have  seen  that  some  enzymes,  especially  certain 
oxidizing  enzymes  and  the  proteolytic  enzymes  derived  from  certain 
bacteria  (Bacillus  prodigiosus,  for  example)  will  withstand  the  tem- 
perature of  boiling  water.  Two  vegetable  proteolytic  enzymes, 
namely  the  Papain  from  the  pawpaw,  or  fruit  of  Carica  papaya  and  the 
Bromelin  in  pineapples  act  best  at  about  60°  C.  Generally  speaking, 
the  more  nearly  neutral  the  solution  of  the  enzyme  and  the  higher  the 
concentration  of  substrate  it  contains,  the  higher  is  the  optimal  tem- 
perature. It  would  therefore  appear  that  acids  and  alkalies  accelerate 
the  inactivation  of  enzymes  and  that  their  substrates  protect  them, 
probably  owing  to  the  fact  that  they  combine  with  them. 

Exposure  of  an  enzyme  to  moderately  high  temperatures,  for  example 
60°  C.  for  some  hours  generally  results,  not  only  in  loss  of  hydrolyzing 
power,  but  in  the  acquirement  of  a  power  to  inhibit  the  very  hydrolysis 
which  the  active  enzyme  normally  accelerates.  This  phenomenon 
has  been  attributed  by  Bayliss  to  the  formation  of  "Zymoids"  which, 
he  believes,  combine  with  the  enzymes  from  which  they  are  derived 
to  form  an  inactive  compound.  In  some  instances,  however,  it 
appears  that  the  retarding  influence  of  heated  enzymes  arises  not  so 


INFLUENCE  OF  TEMPERATURE  UPON  ENZYMES 

much  from  inactivation  of  the  unheated  enzyme  as  in  preferential 
acceleration  of  the  resynthesis  of  the  substrate,  thus  opposing  the 
reaction  which  the  unheated  enzyme  accelerates.  If  this  actually 
occurs  then  it  is  evident  that  a  shift  in  the  equilibrium  of  the  reaction : 

Substrate  +  water  ^  products  of  hydrolysis 

must  be  brought  about  by  the  heated  enzyme,  of  such  a  nature  as  to 
increase  the  proportion  of  the  substrate  in  equilibrium  with  its  products. 
Such  a  shift  of  equilibrium  would,  of  course,  necessitate  a  consumption 
of  energy  and  a  corresponding  alteration  in  the  material  bringing  it 
about,  i.  e.,  in  the  heated  enzyme. 

When  heated  enzymes  are  allowed  to  stand  in  aqueous  solution  at 
room-temperatures  they  may  undergo  spontaneous  Reactivation.  This 
phenomenon  has  been  more  especially  studied  in  connection  with  the 
Oxidizing  Ferments;  but  Gramentzki  has  observed  a  similar  phenomenon 
in  heated  solutions  of  Diastase,  Invertase  and  pancreatic  Trypsin. 
The  following  are  illustrative  data  obtained  with  the  commercial 
starch-splitting  enzyme  Taka-diastase,  obtained  from  Aspergillus 
oryzGB.  The  enzyme-solution  was  heated  to  95°  and  then  immediately 
cooled  and  allowed  to  stand  at  room  temperatures. 

REACTIVATION  OF  HEAT-INACTIVATED  TAKA-DIASTASE  (GRAMENTZKI). 

Solution  tested.  Hydrolyzing  power. 

Unheated  enzyme '. 12.0 

Heated,  immediately  after  cooling 0.6 

Heated,  twenty-five  minutes  after  cooljng 4.2 

Heated,  seventy-five  minutes  after  cooling 5.5 

Heated,  six  hours  after  cooling 8.2 

Heated,  five  days  after  cooling 12.0 

The  accelerative  influence  of  raising  the  temperature  upon  the 
hydrolysis  of  the  substrate  by  an  enzyme  has  been  frequently  investi- 
gated quantitatively,  and  it  has  been  found  that  the  relationship 
between  the  temperature  and  the  velocity  of  hydrolysis  is  that  which 
commonly  pertains  in  chemical  reactions.  It  may  be  expressed  as 
follows : 


to\ 

f 


-i-      =  e  2   \       tito 
V0 

where  "\ri"  is  the  velocity  of  the  hydrolysis  at  the  temperature  "ti;" 
"v0"  is  the  velocity  of  hydrolysis  at  the  temperature  "to;"  "e"  is 

the  base  of  the  natural  or  "Napierian"  logarithms  (2.71828 ) 

and  "  n"  is  a  constant  which  is  characteristic  for  the  specific  reaction, 
and  is  expressive  of  the  degree  of  effect  which  temperature  exerts 
upon  it.  The  temperature  is  measured  in  "absolute"  units,  that  is  to 
say  in  degrees  centigrade  above  zero  plus  273°.  The  relationship 
only  holds  good,  however,  so  long  as  the  temperatures  employed  do  not 


216  THE  HYDROLYZING  ENZYMES 

exceed  the  "temperature-optimum/'  otherwise  the  secondary  inactiva- 
tion  of  the  enzyme  introduces  a  disturbing  factor. 

It  is  more  simple,  however,  although  less  accurate,  to  estimate  the 
effect  of  temperature  by  the  change  in  velocity  produced  by  a  rise  of 
10°  C.  It  is  found,  as  a  very  general  rule,  excepting  in  the  case  of 
photochemical  reactions,  that  the  value  of  ^  for  chemical  transforma- 
tions is  of  such  a  magnitude  (10,000  or  over)  that  a  rise  of  10°  at  ordi- 
nary room-  or  incubator-temperatures  doubles  or  more  than  doubles 
the  velocity  of  transformation. 

The  "Temperature-coefficient,"  or  ratio: 

Velocity  at  T     +     10° 
Velocity  at  T° 

for  chemical  reactions  is  therefore  2  or  over,  while  for  purely  physical 
processes,  such  as  changes  in  viscosity  or  capillarity  or  for  photo- 
chemical reactions  the  value  of  the  coefficient  generally  only  slightly 
exceeds  unity  or,  in  the  case  of  capillary  phenomena,  may  be  less  than 
unity. 

The  following  are  illustrative  values  of  "/*"  for  various  hydrolyses 
brought  about  by  enzymes.  For  comparison  the  value  of  n  for  the 
hydrolysis  of  cane-sugar  by  acids  is  included. 

Process.  At 

Hydrolysis  of  cane-sugar  by  acids 25,600 

Hydrolysis  of  cane-sugar  by  invertase 11,000 

Hydrolysis  of  starch  by  amylase 12,300 

Hydrolysis  of  triacetin  by  lipase 16,700 

Hydrolysis  of  egg-albumin  by  pepsin       .      .      .      .      .      .      .      .  15,570 

Hydrolysis  of  casein  by  trypsin 37,500 

Inactivation  of  rennet 90,000 

Inactivation  of  pepsin 75,000 

Inactivation  of  invertase 72,COO 

Inactivation  of  trypsin 62, 000 

On  comparing  these  various  figures  it  will  be  seen  that  the  effect  of 
temperature  upon  enzymatic  hydrolyses  is  of  the  same  general  order 
as  its  effect  upon  other  chemical  reactions.  The  Inactivation  of  an 
enzyme  by  heat,  however,  is  exceptionally  accelerated  by  rise  of 
temperature,  the  coefficients  for  all  inactivations  being  very  much 
higher  than  those  for  the  hydrolyses  which  the  enzymes  accelerate. 
This  accounts  for  the  relative  "steepness"  with  which  the  curve  of 
enzymatic  activity  falls  off  after  the  temperature  has  passed  the  opti- 
mum; at  this  point  the  inactivation  of  the  enzyme  is  very  much 
accelerated  by  a  rise  in  temperature  sufficient  only  to  produce  a  slight 
modification  of  the  velocity  of  the  hydrolysis  which  the  enzyme  is 
accomplishing. 

Enzymes  are  also  inactivated  by  exposure  of  their  solutions  to  Light, 
and  especially  to  the  Ultra-violet  Rays.  The  inactivation  by  ultra- 
violet light  occurs  in  the  absence  of  oxygen,  but  the  visible  rays  of 
light,  especially  in  the  presence  of  fluorescent  dyes  such  as  Eosin,  are 


INFLUENCE  UPON  HYDROLYSES  BY  ENZYMES  217 

also  able  to  inactivate  enzymes  provided  oxygen  be  present.  Evi- 
dently two  different  types  of  change  in  enzymes  may  be  brought  about 
by  light,  involving  different  parts  of  the  spectrum. 

THE  INFLUENCE  OF  REACTION  UPON  HYDROLYSES  BY 
ENZYMES. 

The  great  majority  of  the  enzymes  are  very  decidedly  influenced  by 
the  reaction,  or  H+  or  OH"  ion  concentration  of  the  medium  in  which 
they  act.  For  each  enzyme  or  group  of  enzymes  there  is  a  certain 
range  of  H+  or  OH~  concentrations  within  which  they  work  best  and 
below  or  above  which  their  activity  is  impeded.  The  upper  limit  of 
H+  or  OH~  concentrations  is  set  by  the  destruction  of  the  enzyme 
which  rapidly  occurs  in  solutions  which  are  too  acid  or  alkaline.  The 
factor  which  sets  the  lower  limit  is  not  so  easy  to  perceive  since  the 
stability  of  the  enzymes  in  neutral  solutions  is  often  greater  than  it  is 
in  the  faintly  acid  or  alkaline  solutions  in  which  their  hydrolyzing 
activity  is  most  favorably  displayed. 

The  most  striking  dependence  upon  reaction  is  shown  by  the  Proteo- 
lytic  Enzymes,  Pepsin  and  Trypsin.  Pepsin  acts  best  in  a  faintly  acid, 
while  trypsin  acts  best  in  a  faintly  alkaline  medium.  A  slight  excess 
of  alkali  rapidly  destroys  pepsin,  while  an  even  slighter  acidity  inacti- 
vates trypsin.  Both  of  these  enzymes  will  hydrolyze  proteins  in 
neutral  solutions,  but  their  activity  is  much  inferior  to  that  which  they 
will  display  in  a  medium  of  favorable  reaction. 

So  far  as  pepsin  is  concerned  it  is  not  difficult  to  infer  that  the  need 
for  a  slight  acidity  of  the  medium  arises  from  the  fact  that  a  compound 
of  the  pepsin  with  the  acid  is  formed  which  possesses  much  greater 
proteolytic  power  than  the  uncombined  pepsin.  This  is  evidenced 
by  the  fact  that  not  all  acids  are  equally  efficient  in  promoting  the 
hydrolysis  of  protein  by  pepsin,  there  being  a  marked  specificity  in  the 
relationship  of  pepsin  to  Hydrochloric  Acid.  While  other  acids  will 
accelerate  the  hydrolysis  of  proteins  by  pepsin,  their  accelerative 
influence  is  far  inferior  to  that  of  hydrochloric  acid,  and  the  favoring 
action  of  different  acids,  instead  of  running  parallel  to  their  degree  of 
electrolytic  dissociation,  as  we  should  expect  if  it  were  an  effect  purely 
due  to  hydrogen  ions,  bears,  in  fact,  no  relationship  to  the  "strength," 
i.  e.,  dissociation  of  the  acid.  Lactic  Acid,  for  example,  in  equimolecular 
solutions,  has  a  more  favorable  effect  than  sulphuric  acid. 

In  the  case  of  Trypsin  the  accelerative  action  of  alkalies  runs  strictly 
parallel  to  their  dissociation,  so  that  here  we  are  left  in  doubt  as  to 
whether  the  effect  is  due  to  the  formation  of  a  compound  of  the  trypsin 
with  the  base  employed,  or  whether  the  alkali  does  not,  on  the  con- 
trary, act  as  an  accessory  catalyzer,  so  altering  the  substrate  as  to 
render  it  more  susceptible  to  attack  by  the  enzyme. 

There  are  certain  observations,  however,  which  seem  to  show  that 
in  this  case  also  a  compound  is  formed  of  the  trypsin  and  the  added 


218  THE  HYDROLYZING  ENZYMES 

base  which  exerts  a  much  more  intense  proteolytic  action  than  trypsin 
itself.  For  if  we  follow  the  hydrolysis  by  trypsin,  of  an  alkaline  solu- 
tion of  casein  by  means  of  the  gas-chain  (potentiometer)  so  that  we 
obtain  a  measure  of  the  changes  in  the  actual  hydroxyl  ion  concentra- 
tion as  the  hydrolysis  proceeds,  we  find  that  the  alkalinity  of  the  digest 
progressively  diminishes  at  a  uniform  rate  corresponding  to  the 
formula : 

Velocity      =     k(a    —  x) 

until   a  certain   Critical  Alkalinity    is    reached,    which    lies    in    the 

N 

neighborhood   of   10~6N  or     nnn  nnn,  below  which  the  velocity  of 

1,UUU,UUU 

hydrolysis  diminishes  very  much  more  rapidly  than  Wilhelmy's  law 
would  indicate.  For  a  given  concentration  of  trypsin  the  critical 
reaction  is  exactly  the  same  when  a  basic  protein  such  as  protamine  is 
employed  as  when  the  acid  protein,  casein,  is  the  substrate.  Evi- 
dently, therefore,  this  sudden  falling  off  in  the  velocity  of  hydrolysis 
is  not  due  to  any  relationship  of  the  Substrate  to  the  free  alkali  in  the 
digest,  but  rather  to  a  relationship  of  the  Enzyme  to  the  free  alkali. 
The  result  is,  in  fact,  exactly  what  one  would  expect  to  obtain  if  the 
actual  catalyst  were  a  compound  of  trypsin  with  the  alkali.  The 
concentration  of  the  catalyst  would  remain  constant  at  all  alkalinities 
below  those  destructive  of  the  enzyme,  provided  there  was  a  sufficient 
amount  of  unneutralized  alkali  present  to  combine  with  all  of  the 
trypsin.  Directly  the  concentration  of  free  alkali  fell  below  this  limit, 
however,  the  concentration  of  active  enzyme  would  diminish  in  pro- 
portion to  the  diminution  of  alkalinity,  that  is  to  say,  in  proportion  to 
the  extent  of  hydrolysis,  and  the  velocity  of  hydrolysis  would  fall  off 
correspondingly  rapidly. 

Since  in  these  two  instances  we  have  experimental  ground  for  the 
belief  that  the  favorable  influence  of  dilute  acids  or  alkalies  upon  the 
activity  of  the  enzyme  is  due  to  the  formation  of  compounds  with  the 
enzyme,  we  may  infer  that  the  mechanism  of  the  acceleration  by 
acids  or  alkalies  is  probably  the  same  in  other  cases. 


THE  SPECIFICITY  OF  THE  HYDROLYZING  ENZYMES. 

The  various  enzymes  which  hydrolyze  Disaccharides  and  Glucosides 
are  highly  specific  in  their  action,  that  is  to  say,  a  given  enzyme  will 
hydrolyze  a  particular  disaccharide  or  a  particular  type  of  glucoside 
and  no  other.  A  very  beautiful  example  of  the  specific  relationship 
which  subsists  between  the  structure  of  a  glucoside  and  the  nature 
of  the  enzyme  which  attacks  it  is  that  afforded  by  the  enzymatic 
hydrolysis  of  the  various  Methyl  Glucosides.  Four  of  these  glucosides 
are  known,  namely  a-methyl-1-glucoside,  and  /3-methyl-l-glucoside, 


SPECIFICITY  OF  THE  HYDROLYZING  ENZYMES 


219 


a-methyl-d-glucoside  and  /3-methyl-d-glucoside.    Their  structures  are 
represented  below: 


o   o, 


HCOCHa 

\ 

HCOH\ 
HOCH 


HCX 

I 

HCOH 


CH2OH 

a-methyl-d-glucoside. 


CHsOCH 


CH3OCH 

!\ 

HCOH 


:o   o; 


CH2OH 

a-methyl-1-glucoside. 


HOCH 

|     / 
HC/ 

HCOH 


CH2OH 
/3-methyl-d- 
glucoside. 


HCOCH3 
HOCH 
HCOH 

H 

I 

HOCH 


CH2OH 

/8-methyl-l- 
glucoside. 


Of  these  neither  a-  nor  /3-methyl-l-glucoside  are  acted  upon  by 
enzymes.  The  a-methyl-d-glucoside  is  hydrolyzed  by  the  Maltase  in 
yeast,  but  the  j3-methyl-d-glucoside  is  not  hydrolyzed  by  yeast;  it  is, 
on  the  other  hand,  hydrolyzed  by  the  enzyme  Emulsin  which  is  found 
in  the  kernels  of  stony  fruits  such  as'  the  almond  and  in  the  tissues 
of  the  fungus  Aspergilhis  niger.  But  emulsin  is  without  action  on  the 
a-methyl-d-glucoside. 

Similarly,  Invertase,  which  hydrolyzes  cane-sugar  to  glucose  and 
fructose,  will  not  hydrolyze  maltose;  maltase,  which  hydrolyzes  maltose 
to  two  molecules  of  glucose,  will  not  attack  cane-sugar  or  lactose; 
lactase,  which  hydrolyzes  milk-sugar,  will  not  hydrolyze  maltose  or 
cane-sugar. 

Since  these  various  disaccharides  differ  from  one  another  only  in  the 
arrangement  of  the  various  groups  about  the  central  carbon-skeleton, 
the  high  degree  of  specific  interrelationship  with  the  enzymes  which 
attack  them  which  they  display,  led  Emil  Fischer  to  the  view  frequently 
alluded  to  as  the  Lock-and-key  Hypothesis,  whereby  the  enzyme  is 
supposed  to  possess  a  structure  which  fits  a  particular  disaccharide  or 
glucoside  as  the  grooves  of  a  key  fit  the  wards  of  a  lock.  Indeed  there 
is  no  other  way  in  which  we  can  imagine  a  mechanism  which  will  so 
precisely  pick  out  a  particular  arrangement  of  atoms  and  decompose 
that  one  and  no  other.  The  phenomenon  affords,  in  fact,  a  striking 
confirmation  of  the  view  that  these  enzymes  accomplish  the  hydrolysis 
of  the  disaccharides  through  the  formation  of  intermediate  compounds. 

The  fat-splitting  ferments,  or  Lipases  do  not  exhibit  such  extremely 
preferential  specificity.  Nevertheless  some  measure  of  specificity  is 
displayed  in  certain  instances.  Thus  Dakin  found  that  in  a  mixture  of 
the  menthyl  esters  of  d-  and  1-Mandelic  Acid  the  menthyl-d-mandelate 
is  hydrolyzed  by  pancreas-lipase  much  more  rapidly  than  the  menthyl- 
1-mandelate,  so  that  the  mandelic  acid  which  results  from  the  hydrolysis 
is  strongly  dextrorotatory. 


220  THE  HYDROLYZING  ENZYMES 

Among  the  Proteolytic  Enzymes  we  again  meet  with  a  series  of  specific 
relationships  between  the  enzymes  and  the  substances  which  they 
hydrolyze.  This  specificity  is  not  revealed  when  we  act  upon  Proteins 
with  various  Trypsins  since  every  protein  which  is  soluble  contains 
some  of  the  linkages  which  are  susceptible  to  attack  by  any  given 
trypsin.  Digestion  of  a  protein  wrill  therefore  proceed  with  trypsin 
from  any  source,  and  if  the  linkages  in  the  molecule  which  are  attacked 
differ  with  the  type  of  trypsin  employed,  we  have  at  present  no  certain 
means  of  ascertaining  that  fact  in  so  complicated  a  molecule  as  that  of  a 
protein.  It  was  for  this  reason  that  the  specificity  of  different  trypsins 
was  until  recently  totally  unsuspected,  and  it  was  tacitly  assumed 
that  all  of  the  enzymes  which  hydrolyze  native  proteins  to  amino- 
acids  in  faintly  alkaline  solutions  were  identical.  The  employment  of 
synthetic  Peptides  of  known  structure  and  configuration  as  substrates, 
however,  has  recently  revealed  to  us  a  heretofore  unsuspected  multi- 
plicity of  protein-hydrolyzing  enzymes. 

The  trypsin  in  pancreatic  juice  was  found  by  Fischer  and  Abder- 
halden  to  hydrolyze  certain  synthetic  peptides  while  others  remain 
unattacked.  The  various  peptides  which  they  investigated  were 
distributed  between  these  two  classes  as  follows: 

HYDROLYZED. 

*Alanyl-glycine  *Alanyl-leucyl  glycine 
*Alanyl-alanine  Dialanyl-cystine 

*Leucyl-isoserine  Dileucyl-cystine 

Glycl-1-tyrosine  Tetraglycl-glycine 
Leucyl-1-tyrosine  Triglycl-glycine  ester 

*Alanyl-glycyl-glycine  d-alanyl-d-alanine 

*Leucyl-glycyl-glycine  d-alanyl-1-leucine 

*Glycyl-ieucyl-alanine  1-leucyl-l-leucine 

l-leucyl-d-glutamic  acid 

NOT  HYDROLYZED. 

Glycyl-alanine  Leucyl-proline 

Glycyl-glycine  Diglycyl-glycine 

Leucyl-alanine  Triglycyl-glycine 

Leucyl-glycine  Dileucylglycyl-glycine 

Aminobutyryl-glycine  d-alanyl-1-alanine 

Valyl-glycine  1-alanyl-d-alanine 

Glycyi-phenylalanine  1-leucyl-d-leucine 

d-leucyl-1-leucine 

It  will  be  observed  that  especially  among  the  various  dipeptides  formed 
by  the  union  of  d-  and  1-alanine  with  d-  and  1-leucine  the  specificity  of 
the  enzyme  is  very  strongly  marked.  It  will  be  noted  also  that  mere 
length  of  the  pep  tide-chain  confers  upon  it  susceptibility  to  attack. 
Thus  diglycyl-glycine  and  triglycyl-glycine  were  not  attacked,  while 
tetraglycylglycine  was  hydrolyzed.  The  compounds  marked  with  an 
asterisk  were  racemic,  and  in  every  case  only  one  of  the  optical  anti- 
podes was  attacked,  in  every  case  also  the  isomer  which  was  hydrolyzed 
was  that  which  occurs  in  the  native  proteins. 


SYNTHETIC  ACTION  OF  HYDROLYZING  ENZYMES          221 

The  trypsin  which  is  contained  in  red  blood-corpuscles  was  found  to 
hydrolyze  glycyl-1-tyrosine,  in  this  respect  resembling  the  trypsin  of 
pancreatic  juice.  It  also  hydrolyzed  diglycyl-glycine,  however,  and 
therefore  it  cannot  be  identical  with  the  trypsin  of  pancreatic  juice. 
Blood-serum  will  not  hydrolyze  glycyl-1-tyrosine  and  the  trypsin  which 
it  contains  therefore  differs  both  from  pancreatic  trypsin  and  red-blood- 
corpuscle  trypsin,  yet  it  will  hydrolyze  d-1-alanyl-glycine,  diglycyl- 
glycine  and  tri-glycyl-glycine.  The  existence  of  three  different  trypsins 
is  thus  demonstrated,  and  from  these  and  similar  experiments  we  can 
infer  that  the  variety  of  animal  trypsins  is  very  great  and  possibly 
coextensive  with  the  number  of  different  types  of  tissue  which  may 
comprise  the  body  of  a  multicellular  animal. 

THE  SYNTHETIC  ACTION  OF  HYDROLYZING  ENZYMES. 

When  we  hydrolyze  such  a  substance  as  Ethyl  Butyrate  with  the  aid 
of  a  non-enzymatic  catalyzer,  we  find  that  the  transformation  into 
ethyl  alcohol  and  butyric  acid  is  never  complete,  but  stops  short 
when,  in  dilute  solutions,  about  two- thirds  of  the  ester  is  decomposed. 
No  matter  what  catalyzer  we  may  employ,  or  if  we  allow  spontaneous 
hydrolysis  to  occur,  or  bring  about  hydrolysis  by  means  of  a  fat-split- 
ting enzyme,  the  transformation  comes  to  a  standstill  when  about  one- 
third  of  the  ester  still  remains  undecomposed.1  On  the  other  hand, 
if  we  mix  ethyl  alcohol  and  buytric  acid,  and  by  the  agency  of  cata- 
lyzers or  otherwise,  bring  about  their  combination,  we  will  find  that 
here  also  the  transformation  is  never  complete,  but  that  it  comes  to  a 
standstill  when  about  one-third  of  the  alcohol  and  buytric  acid  have 
combined  to  form  the  ester.  Evidently,  therefore,  from  whichever  end 
of  the  process  we  start  we  reach  a  mixture  of  the  same  composition. 
We  cannot  suppose  that  either  reaction  has  then  ceased  to  occur,  but 
we  can  readily  see  that  in  the  mixture  which  no  longer  alters  in  com- 
position and  is  at  equilibrium  the  forward  and  reverse  actions  are  pro- 
ceeding at  the  same  rate: 

C3H7COOC2HB     +     H2O     ;±     C3H7COOH     +     C2H6OH 

In  a  variety  of  hydrolyses  the  same  phenomenon  is  observed,  but  in 
the  majority  of  instances  the  station  of  equilibrium  lies  further  to  the 
right  or  left  than  in  the  instance  chosen  for  illustration.  Thus  in  the 
hydrolysis  of  cane-sugar  it  lies  so  far  to  the  left  that  at  equilibrium  the 
hydrolysis  is,  so  far  as  all  practicable  measurements  are  concerned, 
absolutely  complete. 

Now  a  true  catalyzer  is,  as  we  have  seen,  not  consumed  at  all  during 
the  progress  of  the  process  which  it  accelerates,  and,  this  being  the 
case,  it  cannot  communicate  any  Energy  to  the  system.  Any  shift  of 
equilibrium  in  a  chemical  reaction  which  absorbs  or  liberates  heat 
must  involve  the  consumption  or  absorption  of  a  quantity  of  energy 
equivalent  to  the  heat  of  reaction.  But  equilibrium,  as  we  have  seen, 

1  The  exact  proportion  depends,  as  we  shall  see,  upon  the  dilution  of  the  solution, 
i.  e.,  upon  the  mass  of  water  in  the  reacting  mixture, 


222  THE  HYDROLYZING  ENZYMES 

occurs  only  when  the  forward  and  reverse  velocities  are  equal,  and 
hence  if  the  forward  reaction,  or  reaction  of  hydrolysis,  is  accelerated 
by  a  catalyzer,  the  reverse  reaction  or  reaction  of  synthesis  must  also 
be  accelerated  and  to  an  exactly  equal  degree.  If,  then,  the  hydrolyz- 
ing  enzyme's  are  analogous  to  other  catalyzers  and  are  not  consumed 
during  the  progress  of  the  reactions  which  they  affect,  they  must 
accelerate  the  resynthesis  of  the  substrate  from  its  products  no  less 
than  the  hydrolysis  of  the  substrate  itself. 

The  prediction,  based  upon  the  above  premises,  made  by  the  Dutch 
chemist  Van't  Hoff  in  1898  that  enzymatic  syntheses  might  prove 
possible  was -verified  experimentally  in  the  same  year  by  Croft  Hill, 
who  succeeded  in  this  manner  in  synthesizing  a  disaccharide  by  acting 
upon  a  highly  concentrated  solution  of  glucose  with  the  enzyme 
Maltase  obtained  from  yeast. 

The  synthetic  disaccharide  was,  very  naturally,  assumed  to  be 
Maltose,  but  further  investigation  showed  that  the  prediction  of 
Van't  Hoff  had  not  been  so  completely  verified  as  was  at  first  supposed, 
for  Emmerling  in  1901  showed  that  the  disaccharide  which  was  actually 
produced  in  Croft  Hill's  experiment  was  not  maltose,  or  glucose-a- 
glucoside,  but  a  disaccharide  which  yields  a  predominating  proportion 
of  /3-glucose  on  hydrolysis,  namely  glucose-/3-glucoside,  or  Isomaltose. 
Now  isomaltose  is  not  hydrolyzable  by  maltase,  so  that  the  synthetic 
activity  of  the  enzyme,  instead  of  reversing  the  reaction  of  hydrolysis, 
produces  a  disaccharide  which  it  cannot  hydrolyze.  It  would  appear 
that  a  shift  of  equilibrium  is  actually  occasioned  by  the  enzyme,  but  as 
there  is  no  difference  of  energy-content  between  optical  isomers,  the 
shift  in  the  station  of  equilibrium  caused  by  the  enzyme  is  not,  so  far 
as  the  production  of  isomaltose  instead  of  maltose  is  concerned,  of  such 
a  character  as  to  require  consumption  of  the  enzyme  to  accomplish  the 
liberation  or  absorption  of  energy. 

Isomaltose  is,  however,  hydrolyzable  by  the  enzyme  Emulsin  which 
occurs  in  different  situations  from  those  in  which  maltose  is  found-. 
It  became  at  once  a  matter  of  great  interest  to  ascertain  what  synthetic 
products  would  result  from  the  action  of  emulsin  upon  concentrated 
solutions  of  glucose.  This  experiment  was  carried  out  by  E.  F. 
Armstrong,  who  found  that  the  product  resulting  from  the  synthetic 
action  of  emulsin  was  not  isomaltose,  but  Maltose.  Each  enzyme, 
therefore,  synthesiezs  that  enzyme  which  it  cannot  hydrolyze.  These 
relationships  may  be  schematically  represented  by  the  following 
diagram : 

Emulsin 
Maltose  < Glucose 


Glucose » Isomaltose 

Maltase 


SYNTHETIC  ACTION  OF  HYDROLYZING  ENZYMES         223 

Similarly  the  Lactase  in  kephir  yeast  was  found  to  synthesize,  not 
Lactose  which  it  hydrolyzes,  but  Isolactose,  which  it  does  not  hydrolyze. 

Since  the  publication  of  Croft  Hill's  fundamental  observation  a 
great  number  of  enzymatic  syntheses  have  been  accomplished.  Among 
carbohydrates,  substances  resembling  Starch  and  Glycogen  have  been 
synthesized  through  the  action  of  Diastases,  while  Triacetyl  Glucose 
has  been  formed  from  acetic  acid  and  glucose  under  the  influence  of 
pancreas-extract.  Among  the  fats  Ethyl  Butyrate  has  been  synthesized 
from  ethyl  alcohol  and  butyric  acid,  glyceryl  butyrate,  amyl  butyrate, 
methyl  oleate,  glyceryl  triacetate  and  glyceryl  trioleate  have  all  been 
synthesized  from  their  components  through  the  reversed  action  of 
various  Lipases.  In  the  case  of  methyl  oleate  it  has  been  shown  that 
the  pancreas-lipase  employed  to  bring  about  its  synthesis  definitely 
does  not  affect  the  final  equilibrium  which  is  attained,  for  the  proportion 
of  ester  formed  after  a  sufficient  lapse  of  time  is  independent  of  the 
quantity  of  enzyme  employed,  only  the  Speed  with  which  the  equilib- 
rium is  attained  being  affected.  The  following  are  illustrative  data: 

SYNTHESIS  OF  METHYL   OLEATE    (POTTEVIN). 


Quantity  of 
pancreas- 
extract  employed. 

1      

Percentage  of  ester  formed. 

1  day. 
8 

2  days. 
56 
66 
66 
74 

20  days. 
84 
82 
84 
85 

2      

12 

5     

21 

10     . 

....            43 

Quantitative  data  of  this  description  are  very  important  because 
the  whole  question  whether  the  enzymes  act  as  true  catalyzers  or,  on 
the  contrary,  enter  into  and  affect  the  equilibria  of  the  reactions  which 
they  accelerate,  turns  upon  the  question  whether  the  final  proportion 
of  substrate  to  products  is  at  all  influenced  by  the  presence  of  the 
enzyme.  The  results  of  Pottevin  indicate  that  there  is  no  such  influ- 
ence in  the  case  of  pancreas-lipase  synthesizing  methyl  oleate,  because 
if  there  were,  then  doubling  the  amount  of  enzyme  should  double  the 
displacement  of  equilibrium  and  since  no  measurable  effect  upon  the 
equilibrium  results  from  doubling  or  even  multiplying  by  ten  the 
quantity  of  enzyme  employed,  it  follows  that  the  single  unit  of  enzyme 
also  did  not  affect  the  station  of  equilibrium.  Similarly  A.  E.  Taylor 
has  shown  that  the  station  of  equilibrium  in  the  hydrolysis  of  glyceryl 
triacetate  by  lipase  is  exactly  the  same  as  that  which  is  obtained  when 
sulphuric  is  used  as  the  catalyzer.  Such  measurements  are,  however, 
usually  lacking  in  enzyme  studies,  frequently  because  of  the  technical 
difficulty  of  measurements  extending  over  the  long  periods  required  to 
attain  final  equilibrium. 

In  the  case  of  Glyceryl  Trioleate,  which  differs  from  the  glyceride 
studied  by  Taylor  in  being  insoluble  in  water,  H.  C.  Bradley  has 
attained  results  which  point  rather  clearly  toward  a  decided  displace- 
ment of  equilibrium  by  the  enzyme,  for  he  finds  it  impossible  to  procure 


224  THE  HYDROLYZING  ENZYMES 

any  appreciable  synthesis  of  Triolein  from  oleic  acid  and  glycerol  in  the 
presence  of  fifty  per  cent,  of  water,  although  when  we  start  with 
triolein  in  this  proportion  of  water  an  appreciable  amount  of  triolein 
remains  unhydrolyzed  at  the  end  of  prolonged  hydrolysis.  The 
presence  of  the  enzyme  in  this  case  appears  to  selectively  accelerate 
hydrolysis,  and  if  this  is  the  case  then  the  enzyme  must  be  consumed  in 
the  process. 

Among  the  proteins  the  synthesis  of  a  protamine,  Salmine,  from  a 
concentrated  solution  of  its  digestion-products  has  been  accomplished 
by  A.  E.  Taylor,  who  employed  an  unusually  stable  Trypsin  obtained 
by  extracting  the  liver  of  a  mollusc  (Schizothoerus  nuttalli)  with  glycerol, 
and  adding  this  in  very  large  amounts  to  a  saturated  solution  of  the 
amino-acids  which  finally  result  from  the  hydrolysis  of  protamine. 
Only  a  small  proportion,  about  one-half  of  a  per  cent.,  of  the  original 
protein  was  recovered  and  that  only  after  a  lapse  of  five  months.  The 
identity  of  the  synthetic  protein  with  salmine  was  deduced  from 
analysis  and  general  physical  behavior. 

When  Sodium  Caseinate  in  neutral  solution  is  subjected  to  hydrolysis 
by  Pepsin  a  group  of  infraproteins  results,  which  are  collectively  termed 
Paranucleins,  and  which  subsequently  undergo  further  hydrolysis, 
with  the  production  of  proteoses  and  peptones.  The  paranucleins 
resemble  casein  in  being  soluble  in  dilute  alkalies  and  precipitable  by 
acetic  acid,  but  are  less  soluble  in  dilute  mineral  acids  than  casein 
itself.  When  to  the  concentrated  solution  obtained  by  evaporating 
down  the  final  products  of  the  prolonged  peptic  hydrolysis  of  casein, 
a  very  large  proportion  of  fresh  pepsin  is  added,  after  a  comparatively 
brief  period  (forty-eight  hours)  at  40°  C.  a  precipitate  is  formed  in 
the  mixture  which  appears  to  be  identical  with  paranuclein.  In  this 
case  the  identity  has  been  confirmed  by  immunological  methods.  The 
antiserum  to  casein  or  paranuclein  produced  by  repeatedly  injecting 
these  substances  into  the  circulation  of  rabbits  yields  no  precipitate 
either  with  the  products  of  the  complete  peptic  hydrolysis  of  casein  or 
with  pepsin,  but  it  does  yield  a  precipitate  with  the  synthetic  para- 
nuclein obtained  in  the  manner  outlined,  and  this  precipitate  binds 
the  antibodies  to  casein  and  paranuclein  which  the  serum  contained. 
Since  the  Antibodies  which  appear  in  the  circulation  of  animals  as  a 
result  of  immunizing  them  against  proteins  are  in  the  highest  degree 
specific,  yielding  precipitates  only  with  the  protein  employed  in  the 
immunization  or  with  inf  raprotein  derived  from  it  by  partial  hydrolysis 
the  synthetic  product  may  be  considered  to  be  thus  clearly  identified 
with  the  infraproteins  which  are  the  first  cleavage-products  of  casein. 

In  this  case,  however,  rather  definite  indications  were  obtained  that 
the  Synthesizing  Enzyme  is  not  identical  with  pepsin  itself,  for  it  proved 
possible  to  bring  about  the  synthesis  a  great  deal  more  rapidly  at  70°  C. 
than  at  40°  C.,  while  the  hydrolyzing  activity  of  pepsin  is  inhibited 
altogether  at  this  temperature.  Moreover  not  every  preparation  of 
pepsin  will  yield  the  synthesis.  It  has  been  suggested  that  the  active 


SYNTHETIC  ACTION  OF  HYDROLYZING  ENZYMES          225 

agent  in  accomplishing  the  synthesis  is  a  modification  of  pepsin,  arising 
from  it  by  loss  of  water : 

Hydrolyzing  Pepsin  ^  Synthesizing  Pepsin  +  H2O 

and  that  in  bringing  about  the  hydrolysis  of  protein  the  hydrolyzing 
form  may  partially  lose  water  and  be  transformed  into  the  synthesizing 
form  and  vice  versa,  high  temperatures,  as  usual,  favoring  the  forma- 
tion of  the  anhydride  or  synthesizing  form.  If  this  were  so,  of  course, 
pepsin  would  be  far  from  being  a  true  catalyzer,  since  it  would  enter 
into  and  be  modified  by  the  reactions  which  it  accelerates.  Such 
modification  has,  however,  not  yet  been  shown  to  occur. 

It  will  be  seen,  therefore,  that  while  in  some  instances  the  hydrolyzing 
enzymes  appear  to  act  as  genuine  catalyzers,  in  other  instances  their 
behavior  appears  to  be  inconsistent  with  this  view.  In  any  case,  the 
synthetic  activity  of  the  hydrolyzing  enzymes,  so  far  as  it  has  yet  been 
demonstrable  in  vitro  is  very  inferior  in  point  of  speed  and  completeness 
to  the  synthetic  processes  which  actually  and  continually  occur  in 
living  tissues.  As  we  shall  see,  glucose  is  converted  into  glycogen 
almost  as  rapidly  as  it  can  be  absorbed  and  transported,  dissolved  in 
minute  concentration  in  the.  blood,  to  the  cells  of  the  liver.  Fat  is 
synthesized  from  glycerol  and  fatty  acids  in  the  intestinal  mucosa 
within  a  few  moments  after  absorption,  there  are  strong  reasons  for  the 
belief  that  the  synthesis  of  protein  from  amino-acids  in  the  tissues  is 
not  much  less  rapid.  The  contrast  betweea  these  phenomena  and  the 
prolonged  periods  of  action  and  high  concentrations  both  of  the  enzyme 
and  the  products  required  to  resynthesize  the  substrate  by  a  hydrolyzing 
enzyme,  and  the  fragmentary  yield  which  results,  point  very  strongly 
to  the  existence,  in  living  tissues  of  a  decisively  different  synthesizing 
mechanism  to  that  involved  in  the  reversed  action  of  catalyzers.  Only 
two  alternatives  appear  to  be  open  to  us  in  interpreting  this  dispro- 
portion. Either  the  tissues  employ  enzymes  which  selectively  accele- 
rate syntheses  and  therefore  are  consumed  by  the  syntheses  which  they 
accomplish,  or  else  the  syntheses  in  living  tissues,  composed  though 
they  are  of  over  eighty  per  cent,  of  water,  take  place  under  conditions 
approximating  to  almost  complete  desiccation.  This  latter  alternative 
is  not  so  inconceivable  as  it  might  appear,  because  the  reactions  in 
question  may  possibly  take  place  at  the  surface  of  lipoid  granules 
which  are  emulsified  in  the  protoplasm,  but  which,  being  insoluble  in 
water,  afford  a  medium  which  is  almost  water-free.  The  most  serious 
difficulty  attaching  to  this  view,  however,  is  that  so  many  of  the 
products  so  rapidly  synthesized  in  living  tissues  are  as  insoluble  in 
oils  as  water  is  itself,  for  example  the  proteins.  On  the  other  hand  if 
the  former  alternative  be  adopted  we  are  faced  with  the  difficulty  that 
the  hypothetical  synthesizing  enzymes  have  never  been  obtained  apart 
from  living  tissue,  so  that  either  their  action  is  intimately  bound  up 
with  the  uninjured  Structure  of  the  protoplasm,  or  else  we  have  not  yet 
hit  upon  the  right  methods  of  extracting  them  from  living  tissues  and 
conserving  their  synthetic  activity.  ' 
15 


226  THE  HYDROLYZING  ENZYMES 


ANTIENZYMES. 

Like  the  proteins  and  the  poisonous  products  of  bacterial  metabolism, 
the  various  enzymes,  when  injected  into  the  circulation  of  living 
organisms,  give  rise  to  specific  Antibodies,  or  substances  in  the  circula- 
tion of  the  immunized  animal  which  combine  with  the  enzyme  which 
has  been  injected. 

The  Antienzymes  thus  produced  are  highly  specific  and  bind  only 
the  enzyme  employed  for  immunization.  Normal  blood,  however, 
contains  appreciable  amounts  of  antitrypsin  and  also  of  antirennet, 
which  latter,  however,  is  stated  not  to  be  identical  with  the  antirennet 
produced  by  immunization.  The  antienzymes  appear  as  a  rule  to  be 
very  resistant  to  heat,  withstanding  for  some  time  a  temperature  of 
70°  without  losing  their  power  of  inhibiting  digestion  of  the  enzymes 
which  they  bind. 

Antipepsin  and  Antitrypsin  also  occur  in  notable  quantities  in  the 
tissues  of  Intestinal  Worms,  and  it  is  to  this  that  their  immunity  to 
digestion  is  attributed.  The  immunity  of  the  tissues  of  the  stomach 
to  digestion  by  the  gastric  juice  which  they  produce,  and  of  the  tissues 
of  the  intestine  to  digestion  by  pancreatic  juice  is  similarly  attributed 
to  the  normal  presence,  in  these  tissues,  of  antienzymes. 

The  antigenic  property  of  the  enzymes  rather  strongly  points  toward 
their  ultimate  protein  nature,  for  up  to  the  present  no  substance  has 
been  found  to  produce  antibodies  on  injection  which  has  not  been  a 
protein,  or  a  substance  possibly  contaminated  by  a  protein. 

REFERENCES. 
GENERAL: 

Taylor:     Fermentation,  Univ.  of  California  Pub.  Pathology,  1907,  1,  87. 

Bayliss:     The  Nature  of  Enzyme  Action.     London,   1914. 

Euler:     General  Chemistry  of  the  Enzymes.     Trans,  by  Pope.     New  York,  1912. 

Arrhenius:     Quantitative  Laws  in  Biological  Chemistry.     London,  1915. 

Effront:  Biochemical  Catalysts  in  Life  and  Industry.  Trans,  by  Prescott.  New 
York,  1917. 

Robertson:     The  Physical  Chemistry  of  the  Proteins.     New  York,  1918. 
INFLUENCE  or  TEMPERATURE: 

Arrhenius:     Immunochemistry.     New  York,  1907. 
INFLUENCE  OF  REACTION: 

Kanitz:     Zeit.  f.  physiol.  Chem.,  1902-3,  37,  p.  75. 

Berg  and  Gies:     Jour.  Biol.  Chem.,  1906-7,  2,  p.  489. 

Robertson  and  Schmidt:     Ibid.,  1908-9,  5,  p.  31. 

Loeb:     Biochem.  Zeitsch.,  1909,  19,  p.  534. 
SPECIFICITY  : 

Armstrong:     The  Simple  Carbohydrates  and  the  Glucosides.     London.     2d  ed. 

Fischer  and  Bergell:     Ber.  d.  d.  chem.  Ges.,  1903,  36,  p.  2592;  1904,  37,  p.  3103. 

Dakin:     Jour,  of  Physiol.,  1904,  30,  p.  253;  1905,  32,  p.  199. 

Fischer  and  Abderhalden:  Zeit.  f.  physiol.  Chem.,  1903,  39,  p.  81;  1905,  46,  p.  52; 
1907,  51,  p.  264. 

Abderhalden  and  collaborators  (Bergell,  Rona,  Samuely,  Teruuchi,  Babkin,  Hunter, 
Kautzsch,  Schittenhelm,  Koelker,  Gigon,  Deetjen,  McLester,  Manwaring,  Lussana, 
Rilliet,  Strauss,  Dammhahn,  Pringsheim,  Pincussohn,  Weichardt,  Heise,  Medigre- 
ceanu,  Walther):  Zeit.  f.  physiol.  Chem.,  1903,  39,  p.  9;  1905,  46,  pp.  176  and 
187;  1906,  47.  pp.  159,  346,  359,  391,  466;  1906,  48,  pp.  537  and  557;  1906,  49,  pp. 
1,  21,  26,  31;  1907,  51,  pp.  294,  311,  334;  1907-8,  54,  p.  363;  1908,  55,  pp.  371, 
377,  384,  390,  395,  416;  1908,  57,  p.  332;  1909,  59,  p.  249;  1909,  60,  p.  415;  1909, 
61,  p.  200;  1909,  62,  pp.  120,  136,  145,  243;  1910,  66,  pp.  265,  277;  1910,  68,  p.  471. 


ANTIENZYMES  227 

SYNTHETIC  ACTION: 

Croft  Hill:     Jour.  Chem.  Soc.,  1898,  73,  p.  634. 

Cremer:     Ber.  d.  d.  chem.  Ges.,  1899,  32,  p.  2062. 

Berninzone:     Atti.  del.  soc.  ligi.  di.  scien.  nat.  e.  geograph.,  Genoa,  1900,  11,  p.  327. 

Kastle  and  Loevenhart:     Am.  Chem.  Jour.,  1900,  24,  p.  491. 

Hanriot,  C.  R.:     Acad.  des.  Sci.,  1901,  132,  p.  212. 

Acree  and  Hinkins:     Am.  Chem.  Jour.,  1902,  28,  p.  370. 

Armstrong:     Proc.  Roy.  Soc.  London,  B;  1904,  73,  p.  500. 

Taylor:     Univ.  of  California  Pub.  Pathology,  1904,  1,  p.  33.     Jour.  Biol.  Chem., 
1906-7,  2,  p.  87. 

Bodenstein  and  Dietz:     Zeit.  f.  Elektrochem.,  1906,  12,  p.  605. 

Poltevin:     Bull.  Soc.  China.,  1906,  35,  p.  693.      Ann.  Inst.  Pasteur,  1906,  20,  p.  901. 

Taylor:     Jour.  Biol.  Chem.,  1907,  3,  p.  87. 

Robertson:     Jour.  Biol.  Chem.,  1907,  3,  p.  95;  1908-9,  5,  p.  493. 

Robertson  and  Riddle:     Ibid.,   1911,  9,  p.  295. 

Gay  and  Robertson:     Ibid.,  1912,  12,  p.  233. 

Harden  and  Young:     Biochem.  Jour.,  1913,  7,  p.  630. 
ANTIENZYMES: 

Hildebrandt:     Virchows  Arch.,  1893,  131,  p.  12. 

Achalme:     Ann.  Inst.  Pasteur,  1901,  15,  p.  737. 

Weinland:     Zeit.  f.  Biol.,  1903,  44,  pp.  1  and  45. 

Cathcart:     Jour.  Physiol.,  1904,  31,  p.  497. 

Hedin:     Ibid.,  1905,  32,  p.  390. 

Saiki:     Jour.  Biol.  Chem.,  1907,  3,  p.  395. 

Robertson  and  Hanson:     Jour.  Immunology,  1918,  3,  p.  131. 


CHAPTER  XI. 

THE  DIGESTION  AND  ASSIMILATION  OF  THE 
FOODSTUFFS. 

THE  DIGESTION  OF  THE  CARBOHYDRATES. 

The  Starch  in  our  diet  is  converted  by  cooking  into  "soluble  starch" 
which  is  much  more  readily  hydrolyzed  by  the  starch-splitting  enzymes 
or  Amylase1  than  the  uncooked  material.  The  first  enzyme  to  en- 
counter the  foodstuffs  upon  their  introduction  into  the  alimentary 
canal  is  the  amylase  or  Ptyalin  of  saliva.  This  enzyme  energetically 
hydrolyzes  the  starch  to  maltose,  and  it  is  for  this  reason  that  starch, 
when  it  is  held  in  the  mouth,  presently  begins  to  taste  sweet. 

There  is  no  amylase  in  the  Gastric  Juice,  but  nevertheless  the  diges- 
tion of  starch  or  glycogen  continues  for  some  time  in  the  stomach, 
because  the  optimum  reaction  for  amylase  is  a  very  faint  acidity.  The 
gastric  juice  itself  is  strongly  acid,  in  fact,  far  too  acid  to  permit  the 
action  of  amylase  if  this  enzyme  were  received  directly  into  unneutral- 
ised  and  undiluted  gastric  juice.  But  various  constituents  of  the  diet, 
and  especially  the  proteins,  combine  with  the  Hydrochloric  Acid  of  the 
gastric  juice  and  partially  neutralize  it,  so  that  the  contents  of  the 
stomach  during  the  partaking  of  a  meal  and  the  earlier  periods  of 
digestion  are  either  neutral  or  only  faintly  acid.  Long  before  the 
acidity  of  the  gastric  contents  approaches  that  of  pure  gastric  juice, 
the  pyloric  sphincter  opens  and  permits  the  passage  of  the  semi- 
digested  foodstuffs  in  small  portions  at  a  time  into  the  lumen  of  the 
small  intestine. 

The  maltose  which  is  thus  formed  by  the  digestion  of  starch  is  not 
normally  absorbed  from  the  stomach  either  as  such,  or  in  the  form 
of  its  further  cleavage-product,  glucose.  Under  normal  conditions 
there  is  little  or  no  absorption  of  maltose  or  other  sugars  from  the 
stomach.  If  the  pylorus  be  ligated,  some  absorption  of  sugar  will 
then  be  found  to  occur,  but  only  under  conditions  involving  abnormal 
dilatation.  No  carbohydrate-splitting  enzymes  are  found  in  the 
gastric  juice  of  man.  It  is  stated  that  Lactase  may  often  be  found  in 
the  gastric  juice  of  the  calf,  but  not  in  the  adult  animal. 

After  the  foodstuffs  have  remained  in  the  stomach  for  a  sufficiently 
long  period  to  allow  the  Chyme  to  become  faintly  acid  through  admix- 
ture with  an  excess  of  gastric  juice,  the  pyloric  sphincter  opens  and 
permits  the  passage  of  the  chyme  into  the  upper  part  of  the  small 

1  The  amylases  are  frequently  referred  to  as  Diastases.  In  French  scientific  litera- 
ture the  word  "Diastase"  is  used  as  a  generic  term  to  include  all  types  of  enzymes. 


DIGESTION  OF  THE  CARBOHYDRATES  229 

intestine.  Here  the  foodstuffs  are  very  soon  met  by  the  alkaline 
Pancreatic  Juice,  which  reaches  the  intestine,  in  man,  through  the 
common  duct  of  the  liver  and  the  pancreas. 

The  pancreatic  juice  contains  an  Amylase  which  completes  the  work 
of  the  salivary  amylase  and  furthermore,  a  Maltase  which  converts 
the  maltose,  derived  by  the  action  of  amylase  from  starch,  into  Glucose. 
The  glucose  which  is  thus  formed  is  very  rapidly  absorbed  into  the 
portal  circulation,  and  carried  to  the  liver  where  it  is  converted  into 
Glycogen.  The  rapidity  of  this  conversion  is  very  great.  Thus  the 
quantity  of  glucose  derived  from  the  polysaccharides  in  a  single  meal 
may  very  readily  exceed  one  hundred  grams.  Dissolved  in  all  of  the 
blood  in  the  body,  which  cannot  exceed  seven  liters  in  a  man  of  seventy 
kilos,  this  would  give  a  glucose  concentration  of  no  less  than  1.5  per 
cent.  As  a  matter  of  fact  even  at  the  height  of  absorption  during  a 
meal  rich  in  carbohydrates  the  concentration  of  glucose  in  the  blood 
of  a  person  in  normal  health  never  exceeds  one-tenth  of  this.  As 
rapidly,  therefore,  as  the  glucose  is  taken  to  the  liver  by  the  portal 
circulation,  it  is  transformed  into  the  colloidal  anhydride,  glycogen, 
and  held  in  reserve  for  future  consumption. 

When,  however,  an  extraordinary  load  is  thrown  upon  this  mechan- 
ism, by  the  excessive  ingestion  of  diffusible  sugars,  some  slight  degree 
of  Glucohemia  or  excess  of  sugar  in  the  blood  may  nevertheless  occur 
and  in  these  cases  the  glucohemia  is  relieved  by  the  passage  of  sugar 
into  the  urine.  This  type  of  glycosuria  is  known  as  Alimentary  Glyco- 
suria.  The  sugar  which  is  found  in  the  urine  is  usually  Glucose,  but 
when  cane-sugar  or  sweets  made  of  cane-sugar  have  been  ingested  in 
large  quantities,  Levulose  may  also  be  found  in  the  urine,  together 
with  traces  of  unhydrolyzed  cane-sugar.  Lactose  is  somewhat  more 
readily  absorbed  and  excreted  as  such  than  cane-sugar.  If  either  of 
these  sugars  be  injected  intravenously,  they  appear  unaltered  and 
quantitatively  in  the  urine. 

It  is  an  exceedingly  remarkable  fact  that  whereas  amylase  and 
maltase  are  both  present  in  the  digestive  juices,  Lactase  and  Invertase 
are  usually  completely  absent,  or  if  lactase  is  present  its  action  is 
inconspicuous.  We  have  seen  that  the  unaltered  disaccharides,  if 
absorbed  as  such,  are  not  utilized  but  are  as  promptly  as  possible 
ejected  from  the  circulation  by  the  kidneys.  Yet  lactose  is  the  sole 
carbohydrate  nutriment  of  suckling  infants  and  cane-sugar  is  an  exceed- 
ingly important  item  in  the  dietary  of  modern  peoples.  As  a  matter 
of  fact,  although  the  hydrolysis  of  these  disaccharides  cannot  be 
accomplished  to  any  important  extent  by  the  secretions  which  are 
poured  into  the  alimentary  canal  by  the  various  digestive  glands,  yet 
the  consequence  of  their  ingestion  is  actually  the  appearance  of  in- 
creased glucose  in  the  portal  circulation  and  enhanced  storage  of 
glycogen  in  the  liver.  When  partaken  of  in  reasonable  amounts  they 
are  furthermore  fully  utilized  for  maintenance  and  the  production  of 
energy  in  the  body.  At  some  point  during  their  passage  through  the 


230     DIGESTION  AND  ASSIMILATION  OF  THE  FOODSTUFFS 

intestinal  epithelium  they  are  evidently  broken  down  into  simple 
sugars  or  monosaccharides,  but  the  modifications  induced  by  the 
intestinal  epithelium  go  even  further,  for  the  hydrolysis  of  cane-sugar 
yields  Levulose  as  well  as  glucose,  and  the  hydrolysis  of  lactose  yields 
Galactose  as  well  as  glucose.  We  are  compelled  to  assume  that  the 
levulose  and  galactose  fractions  of  these  molecules  are  converted, 
either  in  the  intestinal  mucosa  or  else  in  the  liver  epithelium,  into 
glucose.  In  the  case  of  levulose  this  presents  little  theoretical  difficulty, 
for  the  partial  conversion  of  levulose  into  glucose  can  be  brought 
about  in  vitro  by  the  prolonged  action  of  dilute  alkali.  We  are  ac- 
quainted with  no  mechanisms,  however,  which  will  accomplish  the 
direct  transformation  of  d-galactose  into  d-glucose,  much  less  with 
any  enzyme  which  will  bring  it  about.  That  the  converse  process, 
the  transformation  of  glucose  into  galactose  may  be  brought  about  in 
living  tissues  is  shown  by  the  Glycosuria  which  immediately  succeeds 
extirpation  of  the  mammary  glands  in  milch-cows  and  goats.  The 
sugar  that  appears  in  the  urine  is  glucose,  and  glucose  only,  although 
the  lactose  for  the  manufacture  of  which  the  excess  of  glucose  had 
previously  been  utilized,  is  a  compound  of  glucose  and  galactose.  The 
remarkable  feature  of  this  transformation  is  that  it  involves  disruption 
of  the  oxide-ring  of  glucose  and  its  reformation  upon  the  opposite  side 
of  the  molecule: 

HCOH  HOCH 


HCOH 


[OCH       / 


\  /  HCOH 


°\ 
HOCH   /  \  HOCH 


HC/ 

HCOH  HCOH 


CH2OH  CH2OH 

ot-d-Glucoae.  d-Galactose. 

The  normal  circulating  form  of  hexose  is  therefore  d-Glucose  and  d- 
glucose  only.  Whatever  form  of  hexose  or  polysaccharide  derived 
from  a  hexose  may  be  ingested,  if  it  is  absorbed  at  all,  it  appears  under 
normal  circumstances  as  Glycogen  in  the  liver,  having  either  reached 
the  liver-cells  in  the  form  of  glucose,  or  else  been  transformed  by  them 
into  glucose  as  a  preliminary  step  in  the  formation  of  this  colloidal 
reserve-carbohydrate.  From  the  liver  the  carbohydrate  material  is 
redistributed  over  the  body  as  the  need  arises,  being  broken  down  to 
glucose  again  before  it  makes  its  appearance  in  the  circulation.  The 
determining  factor  which  regulates  the  discharge  of  this  carbohydrate 
reservoir  is  probably  the  concentration  of  glucose  in  the  blood.  This 


DIGESTION  OF  THE  CARBOHYDRATES  23i 

normally  lies  between  0.5  and  0.15  per  cent,  and  we  may  suppose  that 
when  the  consumption  of  carbohydrate  in  distant  tissues  results  in  a 
certain  degree  of  depletion  of  local  stores,  and  of  the  circulating  glucose, 
the  equilibrium  between  the  glycogen  stores  in  the  liver  and  the 
glucose  in  the  fluids  bathing  the  liver-cells  is  disturbed,  and  glycogen 
breaks  down  in  order  to  restore  it. 

(C6HioO5)n       +     nH2O     ->     nC2Hi2O6) 
Glycogen  Glucose 

We  must  suppose  that  Pentoses,  in  so  far  as  they  form  constituents 
of  animal  tissues,  namely  in  the  d-Ribose  radical  of  guanylic  and 
inosinic  acids,  are  derived  from  pentoses  originally  contained  in  the 
diet.  It  will  be  recollected  that  guanylic  and  inosinic  acids  represent 
fragments  derivable  by  partial  hydrolysis  from  vegetable  nucleic  acid, 
and  catalytic  agents  capable  of  bringing  about  this  cleavage  are  found 
widely  distributed  in  animal  tissues  and  tissue  fluids  The  mono- 
nucleotids  are  not  improbably  absorbed  as  such,  the  adenine  mononucle- 
otid  being  transformed  by  direct  deaminization  into  the  corresponding 
hypoxanthine  derivative. 

An  important  proportion  of  the  dietary  of  herbivora,  however, 
is  furnished  by  Pentosans,  or  polysaccharides  derived  from  pentoses. 
We  have  no  evidence  of  the  existence,  either  in  the  digestive  juices  or 
in  the  epithelial  wall  of  the  intestine,  of  any  enzymes  capable  of  trans- 
forming these  substances  into  glucose.  Yet  the  experimental  fact 
remains  that  pentoses  can  be  utilized  by  animals,  and  energy  derived 
from  them  for  the  performance  of  work  and  the  maintenance  of  the 
body.  Whether  they  are  absorbed  and  oxidized  in  the  tissues  as  such 
or  as  glucose  is  not  known,  but  the  administration  of  Rhamnose  to  a 
diabetic  whose  urine  has  been  made  sugar-free  by  the  exclusion  of 
carbohydrates  from  the  diet,  leads  to  reappearance  of  glucose  in  the 
urine. 

The  Celluloses  in  the  dietary  are  indigestible  by  any  of  the  enzymes 
produced  by  the  digestive  glands  or  the  intestinal  epithelium.  Never- 
theless they  are  partially  utilized,  as  much  as  forty  per  cent,  of  young 
and  tender  cellulose,  such  as  that  occurring  in  lettuce,  being  utilizable 
for  the  production  of  energy  by  human  beings.  We  owe  this  ability 
to  the  digestive  activities  of  the  bacteria  which  inhabit  the  lower 
intestine.  The  bacterial  flora  is  particularly  abundant  in  the  lower 
intestine  of  the  herbivora,  and  as  much  as  seventy  per  cent,  of  cellulose 
may  be  dissolved  in  vitro  by  the  intestinal  juices  of  a  horse.  The 
products  of  this  digestion  are  not  monosaccharides,  but  carbon  dioxide, 
methane  and  fatty  acids  of  which  the  latter  only,  of  course,  are  avail- 
able for  nutrient  purposes. 

The  most  important  function  of  the  celluloses  in  the  diet,  however, 
is  that  of  communicating  bulk  to  the  intestinal  contents,  and  promot- 
ing peristalsis  by  affording  a  favorable  consistency  and  volume  for 
propulsion  with  a  minimum  of  muscular  effort.  This  function  of  the 


232     DIGESTION  AND  ASSIMILATION  OF  THE  FOODSTUFFS 

celluloses  is  most  especially  important  in  those  animals,  such  as  the 
herbivora,  which  have  very  long  intestines.  In  man,  however,  too 
large  a  proportion  of  indigestible  carbohydrate,  as  for  example  in  the 
dietary  of  vegetarians,  may  lead  to  incomplete  absorption  of  the 
digestible  foodstuffs  and  promote,  in  this  way,  bacterial  activities  to 
an  undesirable  extent. 

The  Digestion  of  the  Fats. — Very  slight  lipolytic  action  is  exerted  by 
the  Gastric  Juice.  Not  only  is  the  lipase-content  of  gastric  juice  low, 
or  the  lipase  weak  in  action,  but  the  fats,  while  they  remain  in  the 
stomach,  being  insoluble  in  water  and  in  the  form  of  relatively  large 
masses,  present  only  a  limited  surface  of  contact  with  the  gastric 
juice,  and  the  Lipase  which  it  contains  can  hydrolyze  the  fat-masses 
only  at  their  surface.  However,  the  slight  action  which  is  exerted  by 
the  gastric  lipase  is  probably  of  no  little  importance,  for  it  ensures 
that  upon  the  entry  of  the  fats  into  the  upper  part  of  the  small  intestine 
they  contain  a  small  admixture  of  fatty  acid,  which  greatly  promotes 
their  rapid  emulsification  by  the  alkaline  fluids  with  which  they  here 
come  into  contact. 

The  path  of  Absorption  of  the  fats  is  quite  different  from  that  which 
is  followed  by  the  carbohydrate.  Instead  of  passing  into  the  blood- 
stream, after  having  traversed  the  epithelial  lining  of  the  intestine, 
they  are  deflected  into  the  lymphatics  and  carried  thence  into  the 
thoracic  duct.  After  a  meal  rich  in  fats,  the  numerous  small  lymph- 
atic vessels  coming  away  from  the  small  intestine  are  full  of  milky 
fluid  and  stand  out  distinctly  from  the  surrounding  tissues,  by  reason 
of  their  whiteness  and  opacity,  whereas  under  resting  conditions,  when 
digestion  is  not  proceeding,  they  are  transparent  and  difficult  to 
distinguish. 

It  is,  therefore,  possible  to  follow  the  absorption  of  fat  from  the 
small  intestines  by  naked-eye  examination,  and  it  was  in  this  way  that 
Claude  Bernard  in  1846,  discovered  that  the  Pancreatic  Juice  is  essen- 
tial for  the  digestion  and  absorption  of  fats,  for  no  absorption  is 
evidenced  by  the  appearance  of  fat  in  the  lacteals  until  the  foodstuffs 
have  reached  the  point  at  which  the  pancreatic  duct  opens  into  the 
duodenum.  In  man  the  ducts  from  the  liver  and  the  pancreas  join  to 
form  a  common  channel  of  discharge,  in  the  dog  the  two  ducts  enter 
the  intestine  very  close  together,  but  in  the  rabbit  a  considerable 
interval  separates  the  openings  of  the  two  ducts,  the  Bile  from  the 
liver  being  discharged  into  the  intestine  at  a  point  considerably  above 
that  at  which  the  pancreatic  duct  opens.  In  the  space  between  these 
two  ducts  no  absorption  of  fat  whatever  is  to  be  observed  after  a  meal, 
but  immediately  below  the  pancreatic  duct  the  lacteals  are  seen  to  be 
filled  with  emulsified  fat. 

Not  only  the  pancreatic  juice,  but  also  the  bile  is  essential  for  the 
absorption  of  fat,  however,  for  if  by  surgical  procedures  the  bile-duct 
be  made  to  open  into  the  intestine  below,  instead  of  above  the  pan- 
creatic duct,  the  lacteals  in  the  space  between  the  ducts,  notwithstand- 


DIGESTION  OF  THE  CARBOHYDRATES  233 

ing  the  admixture  of  pancreatic  juice  with  the  foodstuffs,  are  again 
seen  to  be  clear  and  free  from  fat,  while  immediately  below  the  new 
point  of  entry  of  the  bile-duct  into  the  intestine  active  absorption 
of  fat  is  evidenced.  Moreover,  partial  or  total  occlusion  of  the  bile- 
ducts  through  catarrhal  conditions  or  tumors  is  not  uncommon,  and 
this  invariably  leads  to  very  defective  absorption  of  fat,  a  large 
proportion  of  unabsorbed  fat  passing  into  the  feces,  even  when  the 
discharge  of  pancreatic  juice  into  the  intestine  has  not  been  interfered 
with  at  all. 

Two  separate  factors  are  therefore  essential  for  the  proper  absorp- 
tion of  fats,  namely  the  bile  and  the  pancreatic  juice.  The  fat-split- 
ting enzyme,  Lipase,  is  contained  in  the  pancreatic  juice  and  not  in  the 
bile.  The  essentiality  of  the  bile  in  this  process  arises  not  from  any 
power  of  digesting  fats  which  it  possesses  itself,  but  from  the  facili- 
tation of  the  digestion  of  fats  by  pancreatic  juice  which  it  brings  about. 

The  fats  differ  from  the  other  nutritive  constituents  of  the  diet  in 
their  insolubility  in  water.  The  enzyme,  lipase,  which  accomplishes 
their  digestion  is,  however,  not  only  soluble  in  water,  but  secreted  and 
poured  into  the  intestine  in  a  watery  medium.  To  secure  contact  of 
these  substances  of  diverse  solubilities  some  special  mechanism  is 
required  and  this  is  supplied  by  the  Emulsification  of  the  fat,  partly 
by  the  alkaline  carbonates  contained  in  the  pancreatic  juice  and  the 
bile,  but  especially  by  the  Bile-salts,  sodium  glycocholate  and  tauro- 
cholate. 

By  the  action  of  alkalies  and  alkaline  salts  upon  partially  hydrolyzed 
fat  containing  a  little  fatty  acid,  Soaps  are  formed,  by  combination 
of  part  of  the  base  in  the  alkaline  salt  with  the  fatty  acid. 

Na2C03          +         CnHssCOOH         =         NaHCOs         +         CnHfcCOONa 
Sodium  carbonate  Stearic  acid  Sodium  bicarbonate  Sodium  stearate. 

The  presence  of  a  small  amount  of  soap  facilitates  the  formation 
of  an  emulsion  of  fat  with  water  because  the  soap  tends  to  collect  in 
a  film  at  the  surfaces  of  the  oil-droplets  and  impedes  their  coalescence 
into  larger  drops.  The  concentration  of  the  soap  at  the  surfaces 
of  the  droplets  is  brought  about  by  the  fact  that  they  lower  the  Sur- 
face-tension of  the  oil-water  interface,  so  that,  a  film  having  once 
been  formed,  if  a  discontinuity  should  appear  in  it,  the  surface  of 
oil  which  is  exposed  will  have  a  greater  tension  than  the  surface  of 
soap  which  covers  the  remainder  of  the  droplet.  The  effect  of  this  is 
to  cause  the  exposed  surface,  where  the  film  is  broken,  to  contract 
more  forcibly  than  the  remainder  and  thus  pull  the  edges  of  the  film 
together  again. 

The  emulsification  of  the  fats  in  the  foodstuffs  is  thus  initiated  by 
the  alkaline  carbonate  in  bile  and  pancreatic  juice,  forming  soaps 
with  the  trace  of  fatty  acid  arising  from  lipolytic  action  of  the  gastric 
juice.  The  emulsifying-power  of  the  soaps  is,  however,  far  inferior 
to  that  of  the  bile-salts,  which  reduce  the  tension  of  the  oil-water 


234     DIGESTION  AMD  ASSIMILATION  OF  THE  FOODSTUFFS 

interface  a  great  deal  more  than  soaps  do,  and  the  alkaline  salts  of  the 
pancreatic  juice,  unaided  by  the  sodium  glycocholate  and  taurocholate 
of  the  bile,  are  unable  to  bring  about  sufficiently  rapid  and  complete 
emulsification  to  permit  digestion  and  absorption  to  occur  with  the 
necessary  rapidity  to  ensure  total  utilization  of  the  fats  in  a  meal. 
Furthermore  the  bile-salts,  in  some  way  which  is  not  yet  fully  under- 
stood but  which  also  probably  arises  from  reduction  of  surface-tensions, 
facilitate  the  Absorption  of  the  fatty  acid  and  glycerol  by  the  intestinal 
epithelium  after  the  digestion  of  the  fat  has  been  completed. 

The  emulsification  of  the  fats  enormously  enhances  the  area  of  fat 
and,  therefore,  the  number  of  fat-molecules  which  are  exposed  simul- 
taneously to  the  action  of  lipase.  Thus  one  cubic  centimeter  of  oil 
floating  upon  the  top  of  water  in  a  test-tube  which  is  one  centimeter  in 
diameter  will  be  in  contact  with  the  water. over  an  area  of  0.785  square 
centimeters.  The  same  volume  of  oil,  broken  up  into  ten  thousand 
droplets  and  distributed  through  the  water  would  expose  a  surface  of 
no  less  than  a  hundred  square  centimeters  to  contact  with  substances 
dissolved  in  the  water.  Hence  the  droplets  formed  by  emulsification 
are  rapidly  eroded  and  finally  consumed  and  converted  into  fatty 
acids  and  glycerol  by  the  Lipase  in  the  pancreatic  juice. 

The  carbohydrate  and  the  proteins  are  broken  up  by  the  digestive 
ferments  and  the  intestinal  epithelium  into  their  simple  constituents, 
and  these  are  absorbed  and  carried  to  the  liver  as  such,  to  be  subse- 
quently distributed  therefrom  over  the  body.  The  fats,  on  the  con- 
trary, reach  the  blood  through  the  lymphatic  circulation  without 
preliminary  elaboration  or  reassortment  by  the  liver.  Corresponding  to 
this  fact  we  find  that  they  are  thrown  into  the  circulation,  not  in  the 
form  of  their  simple  components,  but  in  the  comparatively  elaborate 
form  of  Neutral  Fat.  The  fatty  acid  and  alcoholic  radicals  of  the 
original  fat  are,  in  fact,  quantitatively  recombined  within  the  brief 
period  of  their  passage  through  the  substance  of  the  intestinal  epithe- 
lium, and  the  work  of  digestion  is  completely  undone  again  before  the 
fat  appears  in  the  lacteals. 

It  was  inevitable  that  the  appreciation  of  this  fact  by  investigators 
should  suggest  the  question  whether  and  to  what  extent  the  preliminary 
hydrolysis  of  fats  by  lipase  is  essential.  If  the  hydrolysis  of  fat  is 
completely  reversed  within  so  brief  a  period  and  distance,  is  the  hydrol- 
ysis itself  a  necessary  prerequisite  to  absorption? 

Much  investigation  has  been  devoted  to  this  problem,  and  as  a 
result  we  are  in  possession  of  a  variety  of  results  arising  out  of  different 
methods  of  attack.  These  results  indicate  that  notwithstanding  the 
fact  that  the  hydrolytic  splitting  of  the  fats  is  so  temporary  it  is  never- 
theless essential.  Thus  fats  which  are  not  hydrolyzed  by  lipase,  and 
cholesterol  esters,  waxes  and  hydrocarbon  oils  which  simulate  fats  in 
their  solubilities  and  other  physical  characteristics,  but  are  not  decom- 
posed by  lipase,  are  not  absorbed  to  any  measurable  extent.  Even 
when  vaseline  or  liquid  petrolatum  are  administered  in  emulsions, 


DIGESTION  OF  THE  CARBOHYDRATES  235 

formed  by  mixing  them  with  small  quantities  of  an  emulsifiable  fat 
or  oil,  over  ninety-five  per  cent,  of  the  'hydrocarbon  is  recoverable, 
unaltered,  in  the  feces.  The  same  is  true  of  Lanoline  which  consists 
of  a  mixture  of  cholesterol  esters  of  fatty  acids  and  is  not  saponifiable 
by  lipase.  These  esters  are  not  absorbed;  they  pass  into  the  feces 
unaltered,  although  free  cholesterol  itself  can  be  shown  to  undergo 
absorption. 

Fatty  acids  which  are  normally  foreign  to  animal  tissues  are  absorbed, 
but  only  when  the  fat  is  previously  split  into  its  constituents  by  lipase. 
This  has  been  most  conclusively  shown  by  the  following  very  beautiful 
experiment  of  Bloor's.  Bloor  prepared  the  fatty-acid  compounds  of 
the  polyatomic  alcohols  derivable  from  sugars  by  reduction.  Among 
these  Dilaurate  of  Isomannitol  has  a  high  dextrorotation,  while  the  normal 
body-fats  are  optically  inactive.  On  administering  this  substance  to  dogs 
in  their  food  and  collecting  the  chyle  from  the  thoracic  duct  a  large 
proportion  of  fat  was  obtained  which  yielded  Laurie  Acid  on  hydrolysis. 
Laurie  acid  is»not  found  normally  in  animal  fats,  and  it  must  therefore 
have  been  derived  from  the  isomannitol  dilaurate  which  had  been 
administered.  But  the  fat  obtained  from  the  thoracic  duct  was  also 
Optically  Inactive;  therefore,  it  cannot  have  consisted  of  isomannitol 
dilaurate.  The  accuracy  of  the  method  was  sufficient  to  have  detected 
the  absorption  of  0.5  per  cent,  of  the  isomannitol  dilaurate  which  had 
been  administered.  Not  more  than  this  proportion  therefore  can  have 
been  absorbed  without  previous  hydrolysis  by  the  pancreatic  lipase. 

The  carbohydrates,  and,  as  we  shall  see,  the  proteins  are  absorbed 
in  the  form  of  the  simplest  components  into  which  they  can  be  con- 
verted by  the  hydrolyzing  enzymes,  and  are  distributed  to  the  tissues 
after  having  passed  through  the  liver.  The  fats,  on  the  contrary,  are 
thrown  directly  into  the  circulation  in  a  comparatively  complex  form. 
Corresponding  to  this  difference  in  the  method  of  distribution  we  find 
that  the  composition  of  the  tissue-fats  is  very  much  more  dependent 
'  upon  the  varying  nature  of  the  diet  than  the  tissue-carbohydrates  or 
the  tissue-proteins.  Thus  Erucic  Acid,  C2iH4iCOOH,  is  never  normally 
present  in  the  tissue-fats  of  dogs.  Yet  if  rape-seed  oil,  which  contains 
notable  quantities  of  erucic  acid  glyceride,  be  given  to  starving  dogs, 
this  fatty  acid  may  subsequently  be  isolated  from  their  tissues.  The 
normal  melting-point  of  dog-fat  is  20°  C.  Munk  allowed  a  dog  to  fast 
for  nineteen  days,  until  the  tissues  were  presumably  free  of  reserve- 
stores  of  fat.  The  dog  then  weighed  sixteen  kilograms.  It  was  now 
fed  for  fourteen  days  with  mutton  tallow.  The  weight  of  the  animal 
increased  during  this  period  by  seventeen  per  cent.  On  "trying  out" 
the  tissues  1100  grams  of  fat  were  obtained  and  its  melting-point  was 
40°  C.  The  administration  of  the  fat  of  high  melting-point  had,  under 
these  conditions,  led  to  an  abnormally  high  melting-point  of  the  fats 
laid  up  in  the  tissues. 

The  results  which  we  have  quoted  were  obtained  with  starving 
animals.    Under  normal  conditions,  however,  when  the  tissues  are  not 


236      DIGESTION  AND  ASSIMILATION  OF  THE  FOODSTUFFS 

depleted  of  fat-reserves,  they  are  able  to  exert  some  measure  of  control 
over  the  nature  of  the  fats  which  they  assimilate.  Munk  investigated  a 
patient  who  was  afflicted  with  a  fistula  from  which  it  was  possible  to 
collect  the  chyle  before  it  entered  the  blood-stream.  When  this 
patient  was  fed  upon  a  diet  containing  no  other  fat  than  mutton -fat, 
the  chyle-fat  had  nevertheless  a  lower  melting-point  than  mutton-fat. 
When  the  patient  was  fed  with  no  fat  at  all  but  Cetyl  Palmitate,  which 
melts  at  55°,  the  chyle-fat  was  found  to  melt  at  36°  and  to  consist  of 
a  mixture  of  Glyceryl  Tripalmitate  and  glycerides  of  Oleic  Acid  to  the 
extent  of  fourteen  per  cent,  of  the  mixture,  adjudged  by  the  absorption 
of  iodine  by  the  mixed  fats.  Hence  under  normal  conditions  a  measure 
of  control  is  exerted  by  the  intestinal  wall  itself  and  a  proportion  of 
glycerol  and  oleic  acid  may  be  furnished  to  supplement  the  deficiencies 
of  the  dietary.  This  of  course  becomes  impossible  if  the  glycerides  of 
oleic  acid  which  are  present  in  normal  tissues  have  been  previously 
depleted  by  starvation. 

The  fats  may  also  be  modified  in  the  opposite  direction  and  the 
proportion  of  oleic  acid  glycerides  reduced  during  their  transmission 
through  the  intestinal  epithelium.  Thus  when  Cod-liver  Oil,  which 
contains  a  great  excess  of  unsaturated  fatty  acids,  is  administered  to 
dogs,  the  iodine  number  of  the  fats  after  absorption  is  less  than  that 
of  the  fat  in  the  food. 

The  absorption  of  fat  leads  to  a  temporary  increase  in  the  fat  content 
of  the  blood,  where  it  is  held  in  a  finely  emulsified  condition.  The 
ingestion  of  fat-rich  foods,  as  for  example,  cream,  may  result  in  an 
increase  of  the  fat-content  of  the  blood  to  no  less  than  six  times  the 
normal  concentration  during  the  intervals  between  absorption.  Under 
such  circumstances  the  blood-serum  obtained  by  centrifugalizing 
defibrinated  blood  is  often  cloudy  with  suspended  fat  and  globules  of 
fat  may  not  infrequently  be  found  floating  upon  the  top  of  the  column 
of  fluid  in  the  centrifuge-tube.  Ultimately  the  excess  of  fat  disappears 
from  the  blood,  the  neutral  fats  having  been  built  up  into  the  fatty 
connective  tissues.  The  greater  part  of  the  fat-reserve  is  contained 
in  special  fat-cells,  in  which  the  fat  appears,  at  first  in  small  globules, 
and  later  in  larger  globules  which  coalesce  until  the  accumulation  of 
fat  forces  the  protoplasm  into  the  periphery  of  the  cell,  so  that  it 
presents  an  annular  appearance  on  cross-section,  with  the  flattened 
nucleus  forming  a  slight  thickening  of  the  ring  at  one  side.  Occasion- 
ally such  cells  disintegrate  bodily,  it  being  in  this  way  that  the  solid 
constituents  of  Milk  are  formed  in  the  mammary  glands. 

Upon  allowing  fat-rich  blood  to  stand  in  laboratory-glassware  at 
body-temperatures  a  proportion  of  the  fat  becomes  diffusible.  The 
nature  of  the  change  which  occurs  is  not  yet  understood,  nor  is  it  certain 
whether  or  not  this,  or  a  similar  change  in  the  properties  of  circulating 
fat  precedes  its  absorption  by  the  tissues.  Under  certain  pathological 
conditions,  and  particularly  in  Diabetes,  the  percentage  of  fat  in  the 


DIGESTION  OF  THE  CARBOHYDRATES  237 

blood  is  greatly  increased  above  the  normal,  a  condition  which  is  known 
as  Lipemia.  It  is  stated  that  in  these  cases  the  power  of  the  blood  to 
render  fats  diffusible  is  diminished,  the  excess  of  fat  being  present 
wholly  in  the  emulsified,  or  non-diffusible  condition. 

The  Lecithins  are  very  readily  and  rapidly  split  by  lipase  into  fatty 
acids  and  Glycerophosphoric  Acid.  This  latter  compound,  however,  is 
not  split  by  the  digestive  juices.  It  is  not  known  whether  hydrolysis 
necessarily  precedes  the  absorption  of  the  lecithins.  The  rapidity 
with  which  they  are  split  by  lipase  indicates  that  at  any  rate  a  large 
proportion  of  the  lecithins  must  inevitably  be  hydrolyzed  before 
absorption  can  be  completed.  On  the  other  hand  the  extreme  solu- 
bility of  lecithins  in  solutions  of  bile-salts  encourages  the  view  that 
a  part  at  least  of  these  substances  may  be  absorbed  without  prelimi- 
nary digestion,  and  this  view  is  further  supported  by  the  remarkable 
effects  of  egg-lecithin  upon  the  growth  and  development  of  animals 
and  upon  the  nitrogenous  balance,  when  it  is  administered  by  mouth, 
effects  which,  as  yet,  have  not  proved  possible  to  evoke  by  the  adminis- 
tration of  the  constituent  parts  into  which  it  disintegrates  upon 
hydrolysis.  It  is,  however,  possible  that  these  effects  of  administering 
lecithin  may  be  attributable,  not  to  lecithin  itself,  but  to  impurities 
which  are  commonly  associated  with  crude  preparations  of  lecithin. 

Cholesterol  has  been  definitely  shown  to  be  absorbed  as  such.  If 
an  abnormal  quantity  of  cholesterol  be  administered  by  mouth  to 
animals,  the  excess  is  laid  up  in  certain  tissues,  particularly  those  of 
the  liver,  spleen  and  suprarenal  gland,  and  serious  lesions  may  result 
from  the  accumulation  of  these  deposits.  Certain  organs,  e.  g.,  the 
kidneys,  remain  free  from  cholesterol  deposits  even  when  cholesterol  is 
administered  in  very  great  excess,  but  if  lesions  arise  from  some  other 
cause,  for  example  if  Nephritis  is  induced  by  the  administration  of 
uranium  salts,  then  cholesterol  tends  to  become  deposited  in  the 
injured  tissues.  In  rabbits,  but,  so  far  as  has  yet  been  ascertained,  not 
in  other  species  of  animals,  the  administration  of  excess  of  cholesterol 
is  followed  by  the  formation  of  large  deposits  in  the  intima  of  the 
arterial  walls,  particularly  in  the  wall  of  the  aorta,  leading  to  the 
formation  of  atheromatous  lesions  resembling  those  which  are  observed 
in  cases  of  Arteriosclerosis. 

The  Cholesterol  Esters,  however,  are  not  saponifiable  by  lipase  and 
are  not  absorbed.  Hence  Lanoline,  administered  by  mouth,  is  recover- 
able quantitatively  in  the  feces. 

The  Bile-salts,  which  serve  as  a  vehicle  and  adjunct  in  the  absorption 
of  the  fats,  undergo  a  partial  circulation  in  the  body,  for  after  entering 
the  small  intestine  through  the  channel  of  the  bile,  they  are  partially 
reabsorbed  during  their  passage  with  the  foodstuffs  down  the  intestine. 
Thus  Glycocholic  Acid  is  nearly  absent  from  the  bile  of  carnivora,  but 
on  administering  this  bile-acid  to  carnivorous  animals,  it  appears  in 
important  quantities  in  their  bile. 


238     DIGESTION  AND  ASSIMILATION  OF  THE  FOODSTUFFS 

THE  DIGESTION  OF  THE  PROTEINS. 

Until  a  very  few  years  ago  it  was  generally  held  that  the  proteins 
were  absorbed  from  the  stomach  and  intestine  in  the  form  of  Peptones, 
and,  indeed,  prior  to  the  first  years  of  the  present  century,  it  was  be- 
lieved in  many  quarters  that  not  merely  peptones,  but  even  unaltered 
protein  or  at  least  infraproteins  resulting  from  the  very  earliest  cleav- 
age of  the  native  protein  molecule  might  be  absorbed  without  further 
hydrolysis. 

This  view  of  protein-absorption  stood  in  rather  striking  contrast 
to  our  knowledge  of  the  absorption  of  other  foodstuffs  which  were  at  a 
comparatively  early  date  known  to  undergo  complete  or  nearly  com- 
plete hydrolysis  prior  to  their  absorption.  Moreover,  the  elaborate 
machinery  of  enzymes  for  the  splitting  of  proteins  not  only  to  peptones, 
but  to  amino-acids  which  is  provided  by  the  digestive  organs  would 
appear,  if  the  older  view  were  correct,  to  exist  without  any  necessary 
purpose  or  function.  Considering  these  facts  it  may  appear  strange 
that  so  exceptional  a  view  of  protein-absorption  should  ever  have  gained 
general  acceptance;  but,  as  usual  in  the  historical  development  of 
science,  a  misinterpreted  experiment  furnished  the  foundation  for  an 
extensive  edifice  of  erroneous  hypothesis. 

The  observation  which  led  us  astray  was  the  outcome  of  an  experi- 
ment by  Voit,  who,  in  1869,  showed  that  undigested  proteins,  unmixed 
with  gastric  or  pancreatic  juice,  rapidly  disappear  when  they  are 
introduced  into  a  ligated  loop  of  small  intestine,  while  a  little  later  it 
was  further  found  by  Hofmeister  that  proteoses  and  peptones  similarly 
disappear  when  introduced  into  an  isolated  loop  of  intestine.  The 
latter  of  these  observations  received  its  correct  interpretation  when 
Cohnheim,  in  1901,  showed  that  the  Succus  Entericus  which  is  secreted 
by  the  mucous  membrane  of  the  small  intestine,  contains  an  enzyme, 
Erepsin,  which  hydrolyzes  proteoses  and  peptones  to  amino-acids, 
leaving,  however,  native  proteins  with  the  exceptions  of  casein  and  the 
protamines,  unattacked.  The  disappearance  of  peptone  from  an 
intestinal  loop  is  therefore  accounted  for  by  its  hydrolysis  by  erepsin 
into  amino-acids.  The  disappearance  of  native  proteins  such  as  egg- 
albumin  from  isolated  loops  of  intestine  is,  however,  a  more  difficult 
matter  to  interpret,  and  it  cannot  yet  be  said  to  have  been  completely 
elucidated.  It  is,  however,  certain  that  under  normal  conditions 
unaltered  proteins  never  reach  the  circulation  by  absorption  from  the 
intestine  for  the  following  reasons: 

In  the  first  place,  when  native  proteins  are  injected  into  the  circu- 
lation, a  proportion  of  the  protein  thus  introduced  appears  in  the  urine. 
Evidently  it  is  treated  as  a  foreign  constituent  of  the  blood  and  dis- 
charged, in  so  far  as  that  is  possible,  by  the  kidneys.  Another  portion 
of  the  protein  is  discharged  by  the  kidneys  in  a  non-coagulable  form 
which  is  still,  nevertheless,  a  protein.  At  the  same  time  there  is  a 
marked  increase  of  proteose-like  substances  in  the  blood  and  some 
increase  of  the  urea-output. 


DIGESTION  OF  THE  PROTEINS  239 

To  some  extent,  but  a  very  limited  extent  therefore,  parenterally 
introduced  protein,  that  is,  protein  injected  directly  into  the  circu- 
lation, may  be  utilized  by  the  tissues,  since  a  proportion  of  the  protein 
is  evidently  converted  into  a  normal  product  of  protein  catabolism, 
namely,  urea.  But  it  is  also  evident  that  the  utilization  of  protein 
thus  introduced  is  imperfect,  that  it  is  abnormal  because  the  urine 
contains  protein  which  is  not  the  case  when  proteins  are  absorbed 
from  the  digestive  tract,  and  that  the  utilization  of  the  protein  which 
does  occur  is  preceded  by  hydrolytic  cleavage.  Moreover  it  is  not 
even  certain  that  the  additional  urea-output  which  results  from  the 
injection  of  foreign  proteins  is  due  to  utilization  of  the  protein  itself, 
since  it  has  been  found  by  Mendel  and  Rockwood  that  the  intro- 
duction of  a  foreign  protein,  such  as  Edestin  or  Casein  into  the  cir- 
culation leads  to  a  considerably  more  than  proportionate  increase  of 
the  nitrogenous  secretion,  in  other  words  to  actual  destruction  of 
tissue-proteins. 

In  the  second  place,  the  intravenous  or  subcutaneous  injection  of 
proteins  which  are  foreign  to  the  tissues  of  the  animal  receiving  them, 
results  in  the  production  of  a  variety  of  specific  Antibodies  or  substances 
appearing  in  the  circulation  which  have  the  property  of  precipitating 
or  otherwise  modifying  the  protein  employed  for  injection.  If  the 
injections  are  repeated,  and  successive  injections  are  separated  by 
only  a  few  days  from  one  another,  the  result  after  some  weeks  is  the 
production  of  a  specific  Precipitin  which  circulates  in  the  blood  of  the 
immunized  animal,  so  that  if  the  blood-serum  of  the  animal  be  now 
mixed  with  a  solution  of  the  protein  which  was  employed  for  injection, 
that  protein,  but  no  other,  is  precipitated.  If  a  single  injection  be 
made  and  then  a  second  only  after  a  considerable  interval,  e.  g.,  three 
or  four  weeks,  the  effect  of  the  second  injection  is  to  induce  Anaphy- 
lactic  Shock,  a  condition  which  so  strikingly  resembles  peptone-poison- 
ing that  many  investigators  are  of  the  opinion  that  it  is  due  to  the 
development  in  the  tissues  of  the  sensitized  animal  of  an  enzyme  having 
the  specific  ability  to  rapidly  break  down  the  particular  protein 
employed  and  to  convert  it  into  proteoses  or  peptones. 

Now  it  has  been  shown  that  even  after  the  introduction  of  excessive 
amounts  of  native  protein  into  an  isolated  loop  of  intestine,  although 
the  protein  disappears  and  would  seem  to  have  been  absorbed,  yet  no 
evidence  is  obtainable  of  the  development  of  antibodies  in  the  circula- 
tion of  the  animal  so  treated,  nor  is  there  any  sensitisation,  so  that  a 
second  dose  of  the  protein,  after  a  considerable  interval,  does  not  give 
rise  to  symptoms  of  anaphylactic  shock. 

There  can  be  little  doubt  therefore  that  proteins  are  not  absorbed 
without  previous  hydrolysis,  and  there  is  much  ground  for  supposing 
that  even  that  proportion  of  parenterally  introduced  protein  which  is 
utilized  by  the  tissues,  is  utilized  simply  because  it  has  been  excreted 
into  the  intestine  and  reabsorbed  therefrom  after  digestion. 

The  case  against  the  direct  absorption1  of  peptones  from  the  intestine 


240      DIGESTION  AND  ASSIMILATION  OF  THE  FOODSTUFFS 

is  an  even  stronger  one,  for  a  considerable  proportion  of  the  proteoses 
and  peptones  arising  from  the  incomplete  hydrolysis  of  proteins  are 
definitely  toxic,  and  the  absorption  of  this  type  of  protein  digestion- 
product  would  lead  to  incessant  food-intoxication.  It  is  true  that 
peptones  may  be  absorbed  from  a  ligated  stomach,  and  peptones  and 
proteoses  resulting  from  secondary  degenerative  changes  in  the  intes- 
tinal epithelium  may  be  absorbed  after  prolonged  ligation  of  an  intes- 
tinal loop,  and  may  produce  severe  symptoms  of  peptone  intoxication. 
These,  however,  are  circumstances  not  in  the  least  comparable  with 
those  attending  normal  digestion,  for  a  ligated  and  distended  stomach 
becomes  permeable,  as  we  have  seen,  to  carbohydrates  which  are 
normally  not  absorbed  until  they  reach  the  intestine,  so  that  the 
permeability  of  the  gastric  mucosa  is  evidently  deranged  by  this  proc- 
ess. The  absorption  of  toxic  proteoses  from  an  intestinal  loop  is 
attained  only  after  very  prolonged  ligation,  and  while  this  condition 
may  be  comparable  with  that  prevailing  in  severe  intestinal  stasis, 
it  is  certainly  not  comparable  with  the  normal  phenomena  of  digestion 
and  absorption. 

In  certain  cases,  which  must  be  admitted  to  be  exceptional,  individ- 
uals may  display  phenomena  of  Anaphylaxis  after  the  ingestion  of 
particular  proteins  toward  which  the  individual  has  an  idiosyncrasy. 
Thus,  hyper  sensitiveness  to  the  proteins  of  horse-serum  is  not  unusual 
and  is  responsible  for  the  majority  of  cases  of  Serum-sickness  and  deaths 
arising  from  the  use  of  Diphtheria  Antitoxin  prepared  from  horse-serum. 
It  is  especially  frequently  displayed  by  chronic  asthmatics.  A  smaller 
proportion  of  individuals  are  hypersensitive  to  the  proteins  in  the 
white  of  egg  and  betray  symptoms  of  anaphylactic  shock,  such  as 
Asthma,  when  they  have  partaken  of  eggs.  Others,  again,  are  hyper- 
sensitive to  the  protein  in  strawberries,  others  to  the  proteins  in  shell- 
fish and  so  forth.  In  all  of  these  cases  we  must  assume,  from  the  char- 
acter of  the  symptoms,  that  the  absorption  of  a  proportion  of  unaltered, 
native  protein  is  responsible  for  the  disorder.  The  proportion  of  pro- 
tein absorbed  which  would  suffice  to  account  for  these  effects,  however, 
is  excessively  small.  Thus  Wells  has  shown  that  the  injection  of  such 
a  minute  amount  as  one  millionth  of  a  gram  of  crystallized  egg-albumin 
will  sensitize  a  guinea-pig,  so  that  a  subsequent  intravenous  injection 
of  no  more  than  one-tenth  of  a  milligram  of  the  same  substance  will 
lead  to  fatal  shock.  The  comparative  rarity  of  these  phenomena  of 
protein-intoxication,  and  the  minute  proportion  of  absorption  of 
unaltered  protein  which  would  evidently  suffice  to  evoke  them,  afford 
eloquent  testimony  to  the  difficulty  with  which  native  proteins  and 
peptones  are  absorbed  without  preliminary  digestion. 

We  are  thus  brought  indirectly  and  by  the  exclusion  of  other  possi- 
bilities, to  the  conclusion  that  the  proteins  of  the  diet  must  be  com- 
pletely broken  down  into  Amino-acids  prior  to  their  absorption.  Direct 
evidence  of  the  correctness  of  this  view  has,  however,  been  obtained 
in  recent  years  in  a  variety  of  ways,  of  which  the  following  are  the 
more  important. 


DIGESTION  OF  THE  PROTEINS 


241 


In  the  first  place  it  has  been  ascertained,  thanks  to  the  researches 
of  London,  that  during  the  normal  digestion  of  proteins  in  the  intestine 
large  proportions  of  amino-acids  are  actually  formed,  and  progressively 
absorbed  subsequently  to  their  formation.  The  experiment  consisted 
in  establishing  a  number  of  fistulae  at  intervals  along  the  intestinal 
tract,  so  that  samples  of  the  intestinal  contents  could  be  withdrawn 
and  examined  after  varying  periods  of  intestinal  digestion.  The 
animal  was  fed  with  measured  amounts  of  pure  proteins  and  the  diges- 
tion-products obtained  from  the  successive  sectors  of  the  intestine 
(Fig.  7).  It  was  found  that  these  samples  contained  notable  amounts 
of  amino-acids  and,  moreover,  that  the  relative  proportions  of  the 
amino-acids  arising  from  the  dietary  protein  differed  in  different  sec- 


FIG.  7.— Dog  with  intestinal  and  glandular  fistulse.     (After  London.) 

tions  of  the  intestine.  Thus  when  the  animal  received  the  protein 
Gliadin.  the  duodenum  contained  0.75  grams  of  tyrosine  to  2.5  grams 
of  glutamic  acid;  the  jejunum  contained  only  1.1  grams-  of  tyrosine 
per  20.9  grams  of  glutamic  acid  and  the  ileum  contained  only  a  trace 
of  tyrosine  as  contrasted  with  33  grams  of  glutamic  acid.  Evidently 
therefore,  not  only  are  amino-acids  formed  in  the  normal  intestinal 
digestion  of  proteins,  but  they  are  absorbed  selectively,  e.  g.,  in  the 
particular  case  in  point,  tyrosine  was  absorbed  from  the  intestine  much 
more  rapidly  than  glutamic  acid. 

In  the  second  place  it  has  been  shown  by  Folin  and  Denis,  that  if 

amino-acids  are  introduced  into  a  loop  of  intestine  the  non-protein 

nitrogen  of  the  blood  is  decidedly  increased  during  the  period  that 

absorption  might  be  supposed  to  be  occurring,  and  the  origin  of  this 

16 


242     DIGESTION  AND  ASSIMILATION  OF  THE  FOODSTUFFS 

increase  was  subsequently  demonstrated  by  Abderhalden,  who,  by 
employing  an  enormous  volume,  fifty  liters  of  blood,  succeeded  in 
demonstrating  the  presence  therein  of  the  amino-acids  glycine,  alanine, 
leucine,  valine,  proline,  aspartic  acid,  glutamic  acid,  arginine,  histidine 
and  lysine. 

It  has  furthermore  been  shown  by  Abderhalden,  Henriques  and 
others  that  animals  may  be  maintained  in  perfect  nitrogenous  equilib- 
rium for  prolonged  periods  on  a  diet  containing  no  other  source  of 
nitrogen  than  amino-acids.  It  is  necessary,  however,  to  include  in 
this  diet  all  of  the  amino  acids-which  contribute  to  the  composition  of 
the  various  tissue-proteins.  The  omission  of  Tryptophane,  for  example, 
leads  to  daily  loss  of  weight  and,  in  effect,  to  nitrogen-starvation. 
Nitrogen  equilibrium  and  even  nitrogen  retention,  i.  e.,  accretion  of 
tissue,  was  secured  by  Abderhalden  in  a  dog  to  which  a  diet  was  ad- 
ministered containing  the  following  admixture  of  amino-acids  as  the 
sole  source  of  nitrogen:  Glycocoll  5  grams,  d-alanine  10  grams,  1-serine 
3  grams,  1-cystine  2  grams,  d-valine  5  grams,  1-leucine  10  grams,  d-iso- 
leucine  5  grams,  1-aspartic  acid  5  grams,  d-glutamic  acid  15  grams, 
1-phenylalanine  5  grams,  1-tryosine  5  grams,  1-lysine  5  grams,  d-arginine 
5  grams,  1-proline  10  grams,  1-histidine  5  grams,  1-tryptophane  5  grams. 
The  daily  ration  of  amino  acids  therefore  weighed  100  grams  and  con- 
tained 13.87  grams  of  nitrogen.  It  approximately  resembled  in  com- 
position the  mixture  of  products  which  results  from  the  hydrolysis  of 
the  proteins  of  muscular  tissue. 

We  have  seen  therefore:  (1)  That  amino-acids  are  formed  in  impor- 
tant proportion  in  the  intestinal  digestion  of  proteins.  (2)  That  amino- 
acids  may  be  absorbed  from  the  intestine  and,  (3)  that  amino-acids 
suffice  to  supply  the  nitrogenous  needs  of  the  body.  We  may  infer 
that  the  absorption  of  amino-acids  is  a  normal  and  probably  the  only 
normal  method  whereby  the  materials  for  -the  synthesis  of  proteins 
are  conveyed  to  the  tissues. 

The  difficulty  of  demonstrating  the  presence  of  amino-acids  i,n  the 
blood  after  the  absorption  of  the  digestion-products  of  protein  arises 
from  two  sources:  firstly  the  slowness  of  absorption  and  the  rapidity 
of  circulation,  which  results  in  extreme  dilution  of  the  amino-acid 
products  which  enter  the  portal  venous  system,  and  secondly  the 
rapidity  with  which  the  amino-acids  in  the  blood  are  absorbed  from  it 
by  the  tissues.  The  amino-acids  are  therefore  present  in  the  blood 
even  during  the  height  of  absorption  only  in  very  small  concentrations 
and,  to  add  to  the  difficulty  of  the  problem,  the  blood  is  a  fluid  which 
is  very  rich  in  nitrogenous  substances,  proteins,  which  interfere  to  a 
serious  extent  with  the  chemical  manipulations  which  were  formerly 
necessary  for  the  determination  of  small  concentrations  of  amino-acids. 
In  recent  years  the  development  of  our  technical  knowledge  has 
simplified  and  enhanced  the  accuracy  of  our  methods  of  estimation 
and,  in  particular,  the  development  of  the  nitrous-acid  method  of 
estimating  amino-nitrogen  immediately  enabled  us  to  detect  with 


DIGESTION  OF  THE  PROTEINS  243 

_ 

ease  and  certainty  the  accumulation  of  amino-acids  in  the  blood  which 
accompanies  absorption  after  a  meal  rich  in  protein. 

The  absorption  of  amino-acids  from  the  intestine  and  their  conse- 
quent presence  in  the  blood  has  also  been  very  beautifully  demon- 
strated by  Abel,  employing  his  method  of  Vividiffusion.  This  method 
consists  in  deflecting  a  fraction  of  the  blood-stream  and  causing  it  to 
pass  through  a  series  of  collodion-tubes  before  returning  to  the  general 
circulation.  The  collodion-tubes  are  immersed  in  a  salt  solution  of  the 
same  concentration  as  the  inorganic  salts  in  the  blood,  so  that  dif- 
fusible substances  other  than  inorganic  salts  dialyze  out  of  the  blood 
into  the  saline  solution.  By  renewal  of  the  saline  solution  considerable 
quantities  of  the  diffusible  substances  in  blood  may  be  collected  and, 
among  others,  various  amino-acids  (Fig.  8). 


FIG.  8. — Abel's  vividiffusion  apparatus.     (After  Macleod.) 

It  is  thus  evident  that  the  protein  constituents  of  the  dietary  are 
absorbed  into  the  circulation  in  the  form  of  their  amino-acid  compon- 
ents, some  selection  of  the  amino-acids  transmitted  to  the  blood  being 
exercised  by  the  intestinal  epithelium.  The  question  now  arises, 
where,  and  in  what  way,  these  amino-acid  fragments  are  resynthesized 
into  protein. 

Amino-nitrogen  determinations  show  that  the  excess  of  amino-acids 
which  accumulates  in  the  blood  after  a  meal  very  rapidly  disappears, 
while  coincidently  a  considerable  increase  in  the  free  amino-acids  is 
found  to  have  taken  place  in  the  tissues.  The  amino-acids  are  there- 


244      DIGESTION  AND  ASSIMILATION  OF  THE  FOODSTUFFS 

fore  stored  up  in  the  tissues.  There  is  a  limit,  however,  to  the  capacity 
of  the  tissues  to  retain  amino-acids  and  this  upper  limit  of  capacity 
varies  with  different  tissues.  Thus  the  upper  limit  in  the  case  of 
muscular  tissues  is  about  75  milligrams  of  amino-nitrogen  per  hundred 
grams  of  tissue.  This  characteristic  limit  cannot  be  overstepped  and 
if  the  quantity  of  amino-nitrogen  brought  to  the  tissues  exceeds  their 
Assimilation-limit  the  excess  of  amino-acids  in  the  blood  is  destroyed 
by  Deaminization,  the  nitrogen  being  split  off  in  the  form  of  ammonia 
which  is  converted  by  the  liver  into  Urea.  The  Liver  plays  a  leading 
part  in  this  process  and  continually  and  rapidly  desaturates  itself  by 
deaminization  of  the  free  amino-acids  which  it  contains;  doubtless 
other  tissues  share  this  ability,  but  the  power  of  the  liver  to  deaminize 
amino-acids  is  certainly  in  excess  of  that  of  other  tissues,  because, 
although  other  tissues  do  not  show  any  greater  avidity  than  the  liver 
for  amino-acids,  and  do  not  reach  a  higher  saturation-limit  of  amino- 
nitrogen,  yet  within  a  few  hours  after  the  saturation  of  all  the  tissues 
with  amino-acids  which  succeeds  a  protein-rich  meal,  or  injection  of 
amino-acid  mixture,  the  other  organs  all  contain  more  amino-acid 
material  than  the  liver.  When  we  consider,  also,  that  the  liver  is  an 
exceedingly  bulky  organ,  its  possession  of  a  high  deaminizing  power 
ensures  its  overwhelming  predominance  in  this  process. 

When,  for  any  reason  such  as  that  afforded  by  degenerative  changes, 
the  deaminizing  power  of  the  liver  is  impaired,  this  mechanism  for  dis- 
posing of  undue  excess  of  amino-acids  may  prove  insufficient  and  the 
kidneys  may  assist  by  excreting  unaltered  amino-acids.  If,  at  the  same 
time,  the  introduction  of  amino-acids  into  the  blood  is  unnaturally 
rapid,  as  for  instance  by  rapid  injection  of  large  amounts  of  amino-acids 
in  solution,  the  mechanisms  for  their  disposal  may  prove  to  be  entirely 
inadequate  and  serious  symptoms  of  intoxication,  or  even  death  may 
ensue. 

The  absorption  of  amino-acids  from  the  blood  is  never  complete, 
and  free  amino-acids  are  still  present  in  the  blood  even  when  the 
amino-acid  concentration  in  the  tissues  is  far  below  the  saturation- 
limit.  Evidently,  therefore,  we  are  dealing  here  with  an  equilibrium, 
somewhat  resembling  the  partition  of  a  dissolved  substance  between 
two  immiscible  solvents,  increase  in  the  amino-acid  content  of  the  blood 
leading  to  increase,  up  to  the  saturation-limit,  of  the  amino-acid  con- 
tent of  the  tissues,  while  the  loss  of  tissue-protein  which  occurs  in 
Starvation  indicates  that  diminution  of  the  amino-acid  content  of  the 
blood  may  also  lead  to  desaturation  of  the  tissues  by  the  passage  of 
amino-acids  into  the  blood.  The  same  mechanism,  also,  permits  trans- 
fer of  particular  amino-acids  from  one  tissue  to  another  and  explains 
the  otherwise  surprising  fact  that  certain  tissues,  for  example  malignant 
growths,  may  grow  at  the  expense  of  other  tissues,  and  also,  in  part, 
the  fact  that  the  loss  of  weight  of  the  various  organs  and  tissues  in 
starvation  is  very  unequal,  certain  tissues  losing  very  heavily  while 
others  retain  their  weight  very  nearly  undiminished  until  death  ter- 


DIGESTION  OF  THE  PROTEINS  245 

minates  the  process.  It  will  be  recollected  that  a  similar  equilibrium 
between  the  tissues  and  the  blood  obtains  in  the  case  of  glucose  and  its 
anhydride,  glycogen,  a  subnormal  glucose  concentration  in  the  blood 
leading  to  the  splitting  up  of  glycogen  and  liberation  of  glucose  by  the 
liver  to  replenish  the  blood  and  thereby  the  muscular  tissues  to  which 
it  carries  the  carbohydrate  fuel  which  furnishes  the  energy-equivalent 
of  muscular  work.  In  the  same  way  we  may  suppose  that  the  amino- 
acids  in  the  tissues  stand  in  a  relation  of  equilibrium  to  the  amino-acids 
in  the  blood,  on  the  one  hand,  and  to  the  proteins  of  the  tissue  on  the 
other. 

The  proteins  which  are  found  in  the  various  tissues  of  the  body  are 
highly  specialized  and  characteristic  of  the  tissue-elements  in  which 
they  occur.  The  proteins  in  the  various  Connective  Tissues  are  especi- 
ally diverse  in  their  composition  and  characteristics.  Thus  the  pro- 
teins of  fibrous  tissue  are  extraordinarily  rich  in  glycocoll,  and  those  of 
elastic  tissue  are  especially  rich  in  glycocoll  and  also  in  glutamic  acid. 
Among  other  highly  specialized  proteins  may  be  mentioned  the  keratin 
of  horny  epidermal  tissues  which  is  exceptionally  rich  in  cystine,  the 
protamines  which  are  exceptionally  rich  in  diamino-acids,  and  the 
mucins  which  contain  an  amino-carbohydrate  radical.  In  a  less  degree 
the  proteins  of  every  type  of  tissue  and  cell  betray,  either  in  biological 
or  physical  behavior  or  directly,  in  chemical  composition,  evidence 
of  distinctive  architecture. 

The  question  of  the  locality  of  Protein  Synthesis  has  evoked  a  very 
great  deal  of  discussion  and  prompted  a  variety  of  investigations. 
Arguing  that  only  the  normal  blood-proteins,  the  serum-albumins  and 
serum-globulins  could  be  tolerated  in  the  circulation,  and  assuming 
"that  the  amino-acids  were  not,  as  we  now  know  that  they  are,  absorbed 
as  such,  Abderhalden  supposed  that  the  amino-acids  which  result 
from  digestion  are  synthesized  into  protein  in  the  intestinal  epithelium, 
just  as  the  fatty  acids  and  glycerol  are  synthesized  into  fats  during 
their  passage  through  the  intestinal  wall,  but  with  this  difference, 
namely,  that  whereas  the  fats  which  are  thus  synthesized  bear  a  very 
close  relationship  to  the  fats  which  were  present  in  the  diet,  the  protein 
which  was  presumed  to  be  synthesized  must  be  limited  to  the  blood- 
proteins  characteristic  of  the  species. 

This  hypothesis,  however,  made  it  necessary  to  view  the  process  of 
protein  synthesis  as  a  very  roundabout  and  uneconomical  one,  for 
since  the  proteins  of  the  tissues  differ  so  markedly  from  one  another 
and  also  from  the  blood-proteins,  the  blood-proteins  evidently  could 
not  be  built  up  directly  into  tissue-proteins,  but  must  first  be  broken 
down  in  the  tissues  themselves,  their  amino-acids  resorted  and  rear- 
ranged, and  resynthesized  into  the  characteristic  proteins  of  the  tissue 
in  question.  Thus  the  synthetic  work  of  the  intestine  would  have  to 
be  undone  again  in  each  of  the  tissues.  Moreover  in  many  of  the 
tissues  the  process  of  redegradation  and  resynthesis  would  involve  an 
extraordinary  amount  of  waste  of  ami  no-acid  material.  For  example, 


246      DIGESTION  AND  ASSIMILATION  OF  THE  FOODSTUFFS 

the  proteins  of  connective  tissues  could  not  be  synthesized  at  all  from 
serum-albumin,  because  it  contains  no  glycocoll,  and  even  their  syn- 
thesis, from  serum-globulin  would  involve  a  great  deal  of  wastage, 
because,  whereas  serum-globulin  contains  only  3.5  per  cent,  of  glyco- 
coll,  the  connective-tissue  proteins  contain  about  twenty  per  cent., 
so  that  not  less  than  six  molecules  of  serum-globulin  would  have  to  be 
destroyed  to  build  up  one  molecule  of  connective-tissue  protein,  and 
the  greater  part  of  the  remaining  amino-acids  in  these  six  molecules 
would  not  be  needed.  What,  then,  is  to  become  of  them?  They  might 
be  locally  deaminized,  but  the  predominant  deaminizing  tissue  is  that 
of  the  liver.  In  order  to  reach  the  liver  the  rejected  amino-acids  would 
generally  have  to  travel  thereto  in  the  circulation.  If,  on  the  contrary, 
the  amino-acids  rejected  by  the  connective  tissues  are  utilized  by 
another  tissue,  then  in  order  to  travel  from  the  one  tissue  to  another 
they  must  again  enter  into  the  circulation.  Whichever  hypothesis  we 
adopt  we  are  therefore  compelled  to  revert  to  the  presence  of  amino- 
acids  in  the  blood. 

The  discovery  that  the  amino-acid  products  of  digestion  are  actually 
absorbed  into  the  blood-stream  as  such,  and  are  absorbed  from  the 
blood  by  the  tissues,  removes  these  complexities  from  our  interpre- 
tation of  the  process  of  protein  synthesis.  It  seems  most  reasonable 
to  suppose  that  each  tissue  synthesizes  its  own  individual  proteins, 
and  that  it  is  able  to  utilize  for  this  purpose  all  of  the  amino-acids 
which  it  absorbs  for  the  reason  that  the  characteristic  composition  of 
each  individual  tissue-protein  is  already  determined  by  the  character- 
istic admixture  and  proportion  of  the  various  amino-acids  which  that 
tissue  absorbs  and  holds  in  equilibrium  with  the  blood,  on  the  one 
hand,  and  with  the  tissu  -protein  itself  on  the  other.  The  individual 
characteristics  of  the  proteins  of  the  various  tissues  are  therefore 
determined,  in  ultimate  analysis,  by  the  relative  Permeability  of  the 
tissue  in  question  for  various  amino-acids,  i.  e.,  by  the  relative  ease 
with  which  the  amino-acids  traverse  the  boundaries  which  demarcate 
the  tissue. 

It  is  not  unlikely  that  this  mechanism  of  two-sided  equilibrium  is 
limited  in  its  powers  and  that  it  is  for  this  reason  that  it  is  safeguarded 
or  assisted  by  a  degree  of  preliminary  selection  by  the  Intestinal  Epi- 
thelium. It  will  be  recollected  that  the  experiments  of  London  show 
that  if  a  protein  differing  very  widely  from  animal  tissue-protein  be 
administered,  certain  amino-acids  are  absorbed  selectively.  Thus 
from  Gliadin,  tyrosine  is  absorbed  much  more  rapidly  than  the  glut- 
amic  acid  which  it  contains  in  notable  excess.  The  composition  and 
general  nutritional  standard  of  the  tissues  is  therefore  determined  by 
the  following  interrelated  factors  which  are  severally  in  equilibrium: 

(1)  The  selective  absorptive  activities  of  the  intestinal  epithelium. 

(2)  The  general  average  concentration  of  food-products  in  the  blood, 
i.  e.,  the  abundance  of  the  dietary.     (3)  The  deaminizing  activity  of 


DIGESTION  OF  THE  PROTEINS  247 

the  various  tissues  and  particularly  of  the  liver.  (4)  The  "saturation- 
limit"  of  the  tissues  for  amino-acids.  (5)  The  relative  velocities  of  the 
opposed  processes  of  protein  synthesis  and  degradation  in  the  several 
tissues  of  the  body.  Of  these  factors,  two  main  groups  may  be  recog- 
nized. Absorption  and  deaminization  on  the  one  hand,  determining 
the  abundance  of  nutrient  material  in  the  circulating  medium,  and  the 
excess  or  defect  of  the  velocity  of  synthesis  in  comparison  with  that  of 
degradation  in  the  tissues,  on  the  other,  determining  the  rapidity  with 
which  the  available  nutrients  are  utilized.  The  former  factors  are 
largely  subject  to  environmental  influence,  for  example  that  of  the 
abundance  of  the  dietary.  The  latter  factors  are  individually  char- 
acteristic of  the  organism,  and  in  turn  of  the  several  tissues  of  which 
the  organism  is  composed.  Two  main  groups  of  factors,  therefore, 
contribute  to  determine  the  nutrition,  composition  and  growth  of 
organisms,  an  Environmental  Group  and  an  Internal  Regulatory  Group. 
We  shall  see  when  we  come  to  the  consideration  of  the  problem  of 
Growth  (Chapter  XX),  that  the  diverse  significance  of  these  two 
groups  of  factors  may  very  clearly  be  recognized  in  the  processes  of 
development. 

We  have  seen  that  the  intestinal  digestion  of  proteins  leads  to  the 
production  of  amino-acids,  and  that  these  are  absorbed  into  the  blood- 
stream as  such.  A  considerable  degree  of  preliminary  digestion  of 
proteins  is,  however,  achieved  by  the  Pepsin  in  the  gastric  juice,  and 
the  question  therefore  arises  as  to  whether  any  digestion-products  of 
proteins  are  absorbed  from  the  stomach? 

This  question  may  be  answered  in  the  negative.  We  have  already 
seen  that  under  normal  conditions  neither  carbohydrates  nor  fats 
are  absorbed  from  the  stomach  and,  analogously,  protein  digestion- 
products  are  not  absorbed  from  the  stomach.  It  is  true  that  carbo- 
hydrates may  be  absorbed  from  a  li gated  stomach  and  so,  also,  may 
proteoses  and  peptones,  but  this  constitutes  a  condition  which  is 
nowise  analogous  to  the  conditions  which  pertain  in  normal  digestion. 
The  non-absorption  of  protein  digestion-products  from  the  stomach 
is  in  the  first  place  guaranteed  by  the  fact  that  the  products  of  protein 
hydrolysis  by  pepsin  are  proteoses  and  peptones,  not  amino-acids.  It 
would  not  be  altogether  safe,  of  course,  to  argue  from  the  inability  of 
pepsin  to  digest  peptones  in  vitro  to  a  similar  inability  upon  the  part 
of  the  stomach  in  situ,  but  the  studies  of  London  have  shown  that  the 
production  of  proteoses  and  peptones  is,  in  actuality,  the  main  result 
of  gastric  digestion.  This  investigator  has  established  in  animals  a 
fistula  opening  into  the  intestine  immediately  below  the  pyloric  sphinc- 
ter of  the  stomach.  From  this  fistula  it  is  possible  to  collect  samples 
of  the  stomach  contents  the  moment  after  the  completion  of  gastric 
digestion,  and  their  ejection  into  the  intestine.  The  samples  not  only 
failed  to  contain  any  amino-acids,  but  the  larger  proportion  of  the 
nitrogen  was  present  in  the  form  of  Proteoses,  and  only  a  lesser  proper- 


24S      DIGESTION  AND  ASSIMILATION  OF  THE  FOODSTUFFS 

tion  in  the  form  of  the  further  cleavage-products,  the  Peptones.  The 
following  are  typical  results  obtained: 

Percentage  of  proteoses 

Protein  in  on  completion  of 

the  diet.  gastric  digestion. 

Egg-albumin 72.5 

Gliadin 67.7 

Edestin          60.3 

Casein 59.1 

Gelatin 50.6 

Serum-albumin 46 . 1 

the  remainder  of  the  protein  having  reached  the  peptone-stage  of 
cleavage.  With  varying  quantities .  of  the  same  protein  a  definite 
proportion  of  proteoses  is  always  formed,  as  the  following  results 
illustrate: 

Quantity  of  fdiadin  Percentage  of  proteoses 

in  a  meal.  on  completion  of 

Grams.  gastric  digestion. 

25 80.8 

50 86.1 

75       .      . 86.5 

100 84.9 

The  significance  of  gastric  digestion  lies  in  the  preparatory  work 
which  it  accomplishes  for  the  intestinal  and  pancreatic  enzymes. 
The  hydrolysis  of  proteins  by  Trypsin  is  much  more  rapid  and  complete 
if  the  protein  has  been  subjected  to  preliminary  digestion  by  pepsin, 
and  the  hydrolysis  of  proteins  to  the  peptone  and  proteose  stage, 
furthermore,  converts  the  protein  foodstuffs  into  forms  open  to  attack 
by  the  Erepsin  in  the  succus  entericus.  The  superior  velocity  and 
thoroughness  of  intestinal  protein  digestion  to  the  digestion  of  pro- 
tein in  vitro  by  pancreatic  trypsin  is  attributable  in  large  measure 
to  the  fact  that  the  various  proteolytic  enzymes  act  in  conjunction 
or  succession  upon  the  protein  foodstuffs  in  the  alimentary  canal,  and 
also  to  the  fact  that  the  products  of  digestion  are  removed  almost  as 
rapidly  as  they  are  formed. 

In  addition  to  the  conversion  of  proteins  into  proteoses  and  peptones, 
the  gastric  juice  has  the  special  property  of  converting  the  casein  of 
milk  into  Paracasein.1  Paracasein  has  recently  been  shown  to  be 
derived  from  casein  by  partial  hydrolytic  cleavage,  the  paracasein 
molecule  representing  one-half  the  casein  molecule.  Paracasein 
resembles  casein  very  closely  in  its  general  properties  and  behavior, 
but  its  calcium  salt  is  rendered  insoluble  by  a  very  slight  excess  of 
calcium  ions  at  a  much  lower  temperature  than  the  corresponding  salt 
of  casein  itself.  If  a  sufficiency  of  calcium  chloride,  for  example, 
be  added  to  a  solution  of  calcium  caseinate,  the  protein  salt  will  be 

1  In  British  scientific  literature  these  substances  are  termed,  respectively,  Caseinogen 
and  Casein.  The  word  casein,  therefore,  means  the  unmodified  protein  of  milk,  in 
American  literature,  and  the  infraprotein  derived  therefrom  by  the  action  of  Rennin, 
in  British  literature.  The  American  nomenclature  is  to  be  preferred  because  it  possesses 
the  claim  of  priority,  and  is  that  generally  employed  in  other  languages. 


DIGESTION  OF  THE  PROTEINS  249 

precipitated  at  ordinary  temperatures.  If  a  little  less  calcium  chloride 
be  employed,  it  will  remain  in  solution  at  ordinary  temperatures,  but 
will  form  a  curd  on  elevating  the  temperature.  Even  in  the  absence 
of  free  calcium  ions  a  solution  of  calcium  caseinate  becomes  markedly 
opalescent  on  heating  to  45°  C.  The  calcium  salt  of  paracasein  is, 
however,  for  a  like  concentration  of  free  calcium  ions,  clotted  or  curdled 
at  a  lower  temperature  than  calcium  caseinate.  The  presence  of 
free  calcium  ions  is  therefore  necessary  to  permit  the  clotting  of 
milk  by  gastric  juice  or  extracts  of  the  gastric  mucosa.  They  are  not 
necessary,  however,  for  the  conversion  of  the  casein  into  paracasein, 
which  occurs  just  as  readily  in  a  medium  free  from  calcium  ions  as  in 
one  which  contains  them;  but  visible  evidence  of  the  change  which  has 
occurred  is  lacking  until  calcium  salts  are  added.  Thus  if  excess  of 
ammonium  oxalate  be  added  to  milk,  the  free  calcium  ions  are  removed, 
through  the  formation  of  calcium  oxalate.  The  calcium  combined 
with  the  casein  is  unaffected  because  it  is  not  ionized.  On  adding 
Rennet  (extract  of  gastric  mucosa)  or  gastric  juice  in  small  amount  and 
warming  the  mixture  to  body  temperature  no  visible  change  in  the 
milk  occurs.  The  mixture  may  be  heated  to  boiling  to  destroy  the 
enzyme  without  causing  any  precipitation  or  clotting  of  the  milk,  but  on 
adding  soluble  calcium  salts,  after  cooling,  clotting  of  the  milk  instantly 
occurs.  The  calcium  is  necessary,  therefore,  merely  to  render  the 
product  of  the  enzyme  action  insoluble;  not  to  enable  the  enzyme  to 
act  upon  the  casein.  The  part  played  by  calcium  in  this  process  is 
therefore  sharply  in  contrast  to  the  part  which  it  plays  in  the  coagu- 
lation of  the  blood. 

It  has  long  been  supposed  that  this  change  in  the  properties  of  casein 
which  is  brought  about  by  gastric  juice  is  due  to  a  special  enzyme,  which 
is  termed  Rennin  or  Chymosin.  Evidence  has  accumulated  in  recent 
years,  however,  tending  to  show  that  rennin  is,  in  fact,  identical 
with  Pepsin  and  that  rennet  preparations  which  are  devoid  of  power 
to  digest  proteins  other  than  casein  represent  merely  pepsin,  weakened 
so  greatly  as  to  have  lost  ability  to  hydrolyze  the  majority  of  proteins 
at  any  appreciable  speed.  Thus,  although  pepsin  and  rennin  are 
found  in  a  great  variety  of  situations  both  in  the  animal  and  in  the 
vegetable  kingdoms,  yet  they  are  invariably  found  to  be  associated 
with  cne  another  in  the  same  tissue  or  tissue-fluid.  The  close  relation- 
ship of  rennin  action  to  pepsin  action  is  also  shown  by  the  following 
experiment  of  Morgenroth's.  If  mixtures  of  calcium  caseinate  con- 
taining free  calcium  ions  and  rennet  are  kept  at  low  temperatures 
no  coagulation  occurs,  but  slow  digestion  of  the  casein  (proteose  pro- 
duction) does  occur.  If,  however,  these  mixtures  be  heated  to  20°  C. 
they  clot  immediately.  Thus  the  process  which  underlies  the  clotting, 
has  taken  place  during  the  digestion  which  occurs  at  low  temperatures, 
but  it  cannot  be  visibly  evidenced  by  clotting  until  the  temperature 
is  raised. 


250      DIGESTION  AND  ASSIMILATION  OF  THE  FOODSTUFFS 

THE  TIME-  AND  MASS-RELATIONS  OF  DIGESTION  AND 
ABSORPTION. 

The  Intestine  is  an  extraordinarily  efficient  organ  of  absorption. 
As  much  as  seventy  or  eighty  per  cent,  of  the  total  length  of  the 
jejunum  and  ileum  may  be  removed  and,  provided  fats  be  not  too 
abundant  in  the  diet,  absorption  still  remains  practically  complete. 
If  the  food  contains  a  large  proportion  of  fat,  however,  over  twenty- 
five  per  cent,  of  the  fat  may,  under  these  conditions,  be  discharged 
unabsorbed  in  the  feces  as  contrasted  with  four  or  five  per  cent,  in 
normal  animals  of  similar  kind  and  dimensions. 

The  greater  part  of  absorption  takes  place  in  the  upper  part  of  the 
small  intestine  and  absorption  is  practically  complete  before  the 
contents  of  the  small  intestine  are  discharged  into  the  cecum.  There 
are,  however,  certain  exceptions  to  this  rule,  mainly  furnished  by  dif- 
ficultly digestible  foodstuffs.  Thus  uncooked  white  of  egg  is  digested 
with  great  difficulty  and  as  much  as  seventy  per  cent,  of  this  protein 
may  pass  undigested  and  unabsorbed  into  the  large  intestine  where  it 
may  be  presumed  to  afford  a  favorable  culture  medium  for  putrefactive 
bacteria.  Then,  also,  when  proteins  very  diverse  in  composition  from 
the  normal  tissue-proteins  of  animals,  such  as  certain  vegetable  pro- 
teins, are  partaken  of,  the  selective  absorption  which  occurs  may  result 
in  a  proportion  of  an  amino-acid  which  is  present  in  undue  excess 
remaining  unabsorbed  until  its  passage  into  the  large  intestine. 

The  Stomach,  as  might  be  imagined  from  the  nature  of  the  part  it 
plays  in  digestion,  is  not  essential  to  the  absorption  of  foodstuffs. 
Excision  of  the  stomach  is  followed  by  a  good  utilization  even  of 
proteins,  the  digestion  being  accomplished  by  the  trypsin  of  the  pan- 
creatic juice  and  the  erepsin  of  the  succus  entericus.  Provided  the 
stomach  be  left  in  situ,  moreover,  efficient  digestion  and  absorption  of 
proteins  may  still  continue  when  the  pancreatic  duct  is  ligated,  so 
that  pancreatic  juice  cannot  enter  the  intestine.  In  this  case  digestion 
is  effected  by  erepsin  after  preliminary  cleavage  by  the  pepsin  in  the 
gastric  juice.  The  absorption  of  Fats,  however,  is  very  seriously 
interfered  with  by  the  exclusion  of  pancreatic  juice  from  the  intestine. 

An  important  relationship  subsists  between  the  amount  of  food 
which  is  partaken  of  at  a  meal  and  the  quantity  of  digestive  juices 
secreted  on  the  one  hand,  and  the  time  occupied  in  digestion  on  the 
other  hand.  In  the  case  of  the  Gastric  Juice  the  quantity  of  the 
digestive  fluid  secreted  may  be  estimated  by  forming  a  diverticulum 
or  pocket  in  the  stomach  which  is  connected  with  the  exterior  by 
means  of  a  fistula.  The  juice  secreted  by  this  diverticulum  is  found 
to  be  a  constant  proportion  of  the  fluid  which  is  secreted  by  the  whole 
area  of  the  gastric  mucosa  and  the  total  secretion  of  gastric  juice  during 
digestion  may  therefore  be  estimated  in  terms  of  the  volume  of  the 
secretion  furnished  by  the  diverticulum.  It  has  been  observed  that 
the  quantity  of  gastric  juice  which  is  secreted  during  the  digestion 


RELATIONS  OF  DIGESTION  AND  ABSORPTION  251 

of  a  meal  is  very  closely  proportionate  to  the  quantity  of  food  of 
any  one  kind  which  is  ingested.  The  following  are  results  obtained  by 
Klugine,  the  "calculated"  figures  being  estimated  on  the  assumption 
of  strict  proportionality  between  the  volume  of  secretion  and  the  mass 
of  the  given  type  of  food  which  was  ingested. 

Quantity  Quantity  of  gastric  juice: 

of  food.  Observed.           Calculated. 

Type  of  Food.  grams.  c.c.  c.c. 

Raw  meac       ......*..  400  106  99 

Raw  meat       .      .      .      .-   .'_.-•.      .  200  41  50 

Raw  meat       .      '.....".    ".  100  27  25 

Boiled  meat    .      .      .      .      .      .      .     -.  200  42  42 

Boiled  meat    ........  100  21  21 

Milk I...!.  600  56  53 

Milk 500  41  44 

Milk 200  17  18 

Soup  of  oats  and  meat 600  43  41 

Soup  of  oats  and  meat   .      .      .....  300  20  21 

Meat,  bread  and  milk     .    . .      .      .     -.  800  83  90 

Meat,  bread  and  milk    .      .      .      .      .  400  41  45 

It  is  evident  that  the  calculated  figures  agree  as  closely  as  could  be 
desired  with  those  actually  observed.  Evidently,  then,  there  is  not 
a  constant  amount  of  gastric  juice  secreted  for  each  meal,  but  the 
amount  furnished  is  proportionate,  for  any  one  kind  of  food,  to  the 
mass  ingested.  As  Arrhenius  has  pointed  out,  this  would  appear  at 
first  sight  to  be  an  uneconomical  arrangement,  since  a  very  small 
quantity  of  a  digestive  enzyme  is  capable,  in  time,  of  digesting  a 
very  great  excess  of  foodstuff.  The  length  of  time  required  for  diges- 
tion, however,  if  the  mass  of  enzyme  available  for  each  meal  were  a 
limited,  fixed  quantity,  would  be  so  extremely  variable  that  the 
economy  of  the  tissues  could  not  be  adjusted  to  so  irregular  a  method 
of  furnishing  their  needs.  For  instance  if  about  four  and  a  half  hours 
are  requisite  for  the  gastric  digestion  of  100  grams  of  raw  meat  by  a 
given  amount  of  pepsin  then  it  may  readily  be  calculated  from  the 
Schiitz-Borrissov  Rule,  which  pepsin  obeys,  that  no  less  than  70  hours 
would  be  requisite  for  the  digestion  of  400  grams  and  eighteen  days 
for  the  digestion  of  a  kilogram.  As  a  matter  of  fact,  however,  the 
process  of  gastric  digestion  is  carried  out  in  successive  portions  of 
the  foodstuffs,  a  fresh  supply  of  gastric  juice  being  furnished  for  each 
portion  of  food  that  comes  into  contact  with  the  surface  of  the  gastric 
mucosa.  In  this  way  much  more  rapid  and  uniform  digestion  is 
secured  than  would  otherwise  be  possible. 

The  hydrolysis  of  foodstuffs  in  the  alimentary  canal  appears  to 
follow  the  same  quantitative  laws  as  the  hydrolyses  by  the  corre- 
sponding enzymes  in  vitro.  Thus  Gastric  Digestion  follows  the  Schiitz- 
Borrissov  rule,  while  the  hydrolysis  of  protein  in  the  small  intestine 
by  Pancreatic  Juice  follows  the  monomolecular  logarithmic  formula: 

log  ^r~    =   kt 
which  holds  good  for  the  action  of  this  enzyme  in  glassware. 


252      DIGESTION  AND  ASSIMILATION  OF  THE  FOODSTUFFS 

It  is  a  rather  noteworthy  fact  that  the  Rate  of  Absorption  of  digestion- 
products  from  the  intestine  does  not  appear,  in  so  far  as  it  has  been 
quantitatively  investigated,  to  follow  the  logarithmic  rule,  as  we 
should  expect  if  the  rate  of  absorption  depended  solely  upon  the 
concentration,  i.  e.,  osmotic  pressure,  of  the  substance  undergoing 
absorption.  On  the  contrary,  for  the  absorption  of  glucose  at  all 
events,  a  square-root  rule  seems  to  hold  good,  i.  e.,  the  quantity 
absorbed  in  a  given  time  is  proportional  to  the  square-root  of  the 
concentration  of  the  material  which  is  being  absorbed.  It  is,  however, 
perfectly  evident,  even  apart  from  these  measurements,  that  the  proc- 
ess of  absorption  cannot,  be  purely  a  question  of  the  diffusion  of  sub- 
stances into  and  through  the  wall  of  the  intestine  in  simple  propor- 
tion to  their  osmotic  pressures,  for  otherwise  no  Selective  Absorption 
would  be  possible.  We  have  seen  that  certain  amino-acids  are 
absorbed  preferentially,  others  being  absorbed  with  relative  slowness 
even  when  they  are  present  in  excess.  This  implies  that  besides  the 
forces  of  osmotic  pressure,  phenomena  of  solubility  in  the  absorbing 
tissue-elements  or  of  chemical  affinity  therewith  play  an  important  or 
decisive  part  in  determining  the  relative  rates  of  absorption  and  the 
types  of  material  absorbed. 

From  the  Large  Intestine,  as  we  have  seen,  the  products  of  bacterial 
decomposition  of  foodstuffs  may  be  absorbed,  sometimes  with  physio- 
logically undesirable  results.  A  considerable  Absorption  of  Water  occurs 
here  also.  During  digestion  and  absorption  in  the  stomach  and 
small  intestine  the  contents  of  the  alimentary  canal  retain  a  watery 
consistency  which  is  favorable  to  the  rapidity  of  hydrolysis,  and  to 
the  thorough  admixture  of  the  digestive  secretions  with  the  foodstuffs 
and  the  absorption  of  the  products  of  digestion.  In  the  large  intes- 
tine, however,  a  large  proportion  of  this  water  is  absorbed,  so  that 
the  water-content  of  the  feces  is  normally  considerably  less  than 
that  of  the  contents  of  the  small  intestine.  In  case  the  feces  are 
expelled  with  undue  rapidity,  however,  and  before  the  absorption  of 
water  is  complete,  as  when  a  cathartic  is  administered,  then  the 
feces  have  a  watery  consistency  and  thirst  is  engendered  through 
insufficient  absorption  of  the  water  which  has  been  partaken,  and 
which  has  also  been  furnished  to  the  intestinal  contents  by  the  various 
digestive  fluids. 

Not  only  water  and  products  of  bacterial  action  may  be  absorbed 
from  the  large  intestine,  however,  but  also  foodstuffs  if  they  chance  to 
find  entry  therein  without  previous  absorption.  Thus,  it  is  not  an 
uncommon  procedure  in  medical  practice  to  furnish  nutrition  to 
very  weak  individuals  or  to  persons  who  are  unable  to  swallow,  by 
Rectal  Feeding,  or  the  introduction  of  enemas  containing  fully  hydro- 
lyzed  foodstuffs,  such  as  glucose.  The  substances  thus  administered 
are  found  to  be  absorbed,  and  to  be  normally  utilized  for  the  mainten- 
ance of  the  tissues,  and  the  provision  of  energy. 


RELATIONS  OF  DIGESTION  AND  ABSORPTION  253 


REFERENCES. 

ABSORPTION  OF  CARBOHYDRATES: 

Woodyatt,  Sansum  and  Wilder:     Jour.  Am.  Med.  Assn.,  1915,  65,  p.  2067. 

MacLeod  and  Fulk:     Am.  Jour.  Physiol.,  1917,  42,  p.  193. 
ABSORPTION  OF  FATS: 

Bloor:  Jour.  Biol.  Chem.,  1912,  11,  pp.  141  and  429;  1913,  15,  p.  105;  1913-14, 
16,  p.  517;  1914,  17,  p.  377;  1914,  19,  p.  1;  1915,  22,  p.  133;  1915,  23,  p.  317; 
1916,  24,  p.  447. 

Bloor  and  Knudsen:     Ibid.,  1916,  27,  p.  107. 

Bloor:     Ibid.,  1916,  25,  p.  577;  1916,  26,  p.  417. 
ABSORPTION  OF  PROTEINS: 

Mendel  and  Rockwood:     Am.  Jour.  Physiol.,  1904-5,  12,  p.  336. 

Wells:     Proc.  Soc.  Exp.  Biol.  and  Med.,  1908-9,  6,  p.  1. 

Whipple:     Jour.  Am.  Med.  Assn.,  1915,  65,  p.  476. 

Cathcart:     The  Physiology  of  Protein  Metabolism.     London,  1912. 

Folin  and  Denis:  Jour.  Biol.  Chem.,  1912,  11,  pp.  87  and  161;  1912,  12,  pp.  141, 
and  253. 

Abderhalden:     Zeit.  f.  physiol.  Chem.,  1913,  88,  p.  478. 

Van  Slyke  and  Meyer:  Jour.  Biol.  Chem.,  1912,  12,  p.  399;  1913-14,  16,  pp.  197, 
213  and  231. 

Van  Slyke:     Ibid.,  1913-14,  16,  p.  187. 

Abel,  Rowntree  and  Turner:     Jour.  Pharm.  Exp.  Therap.,  1913-14,  5,  p.  611. 

Turner,  Marshall  and  Lamson:     Ibid.,   1915,  7,  p.   129. 

Underhill:     The  Physiology  of  the  Amino-acids.     Newhaven,  1915. 

Abel:     The  Mellon  Lecture.  Science,  N.  S.,  1915,  43,  p.  135. 
TIME  AND  MASS  RELATION  OF  DIGESTION  AND  ABSORPTION: 

Erlanger  and  Hewlett:     Am.  Jour.  Physiol.,  1901-2,  6,  p.  1. 

Arrhenius:     Quantitative  Laws  in  Biological  Chemistry.     London,  1915. 


PART  II. 

THE  PROPERTIES  OF  PROTOPLASM 


CHAPTER  XII. 

PROPERTIES  CONFERRED  BY  THE  DIFFUSIBLE 
CONSTITUENTS. 

THE  OSMOTIC  PRESSURE  OF  THE  TISSUE  FLUIDS. 

The  diffusible  constituents  of  living  matter  and  of  the  media  which 
bathe  it,  play  a  leading  part  in  determining  the  movements  and  dis- 
tribution of  the  most  abundant  constituent  of  living  cells,  namely 
Water.  Water  is  a  very  essential  constituent  of  protoplasm,  for  a 
variety  of  reasons.  In  the  first  place  it  is  a  solvent  for  the  majority 
of  the  protoplasmic  constituents,  and  thus  permits  their  mobility  and 
promotes,  by  reduction  of  internal  friction  and  cohesion,  the  free 
and  rapid  interplay  of  chemical  reactions  which  characterizes  the 
unstable  equilibria  of  life.  Then,  again,  water  is  the  most  efficient 
ionizing  solvent,  and  thus  permits  electrical  forces  to  come  into  play, 
and  that  notable  increase  in  chemical  reactivity  which  accompanies 
the  ionization  of  dissolved  substances.  The  low  internal  friction  of 
water  permits  the  changes  of  form,  and  rapid  displacements  of  sub- 
stance which  render  the  mobility  of  living  matter  possible.  The  high 
surface-tension  of  water  is  essential  in  the  conservation  of  the  boun- 
daries of  the  cell,  and  their  restoration  after  displacements  due  to 
motion,  and  this,  in  turn,  conserves  the  minute  internal  structures 
of  the  cell.  The  high  specific  heat  of  water  enables  it  to  absorb  a 
great  deal  of  heat  without  increasing  very  greatly  in  temperature  and, 
conversely,  to  part  with  stored-up  heat  without  falling  very  much  in 
temperature.  Sharp  inequalities  of  temperature  which  might  other- 
wise arise  in  living  tissues  are  thus  smoothed  out  by  the  "buffer 
action"  of  the  prevailing  solvent. 

It  is  of  interest  to  consider  the  percentages  of  water  which  are 
contained  in  the  various  tissues  of  the  animal  body.  The  following 
are  illustrative  analyses  cited  after  Hammarsten: 


256  THE  PROPERTIES  OF  PROTOPLASM 

Tissue.  Percentage  of  water 

Fatty  tissue •      •  6-10 

Bone  (extremities  and  skull)        ,      .      .      .      .      ...      .      .      .  14-22 

Bone  (vertebra  and  ribs)       ,      .....      ...»      .      .  16-44 

Tendon .      .      .      .'.'.      .      .      .  56-68 

Brain,  white  substance     .      .      ...      .     ;      .      .      .      ..     .      .  68-70 

Muscular  tissue 75-78 

Thyroid  gland        ...      .      ......      .      .      .      .      .  77-82 

Thymus '     .      .      .  81 

Brain,  gray  substance 82-85 

It  will  immediately  be  noted  that  the  percentage  of  water  is  highest 
in  those  tissues  which  are  undergoing  the  most  rapid  metabolic  changes 
and  which  are  called  upon  to  function,  in  a  chemical  rather  than  a 
structural  manner,  most  rapidly  and  frequently.  The  percentage  of 
water  is  lower  in  adult  than  in  embryonic  -tissue,  and  decreases  with 
advancing  age  of  the  tissues,  and  diminution  of  the  speed  of  metab- 
olism. Living  tissues  which  are  exceptionally  poor  in  water  or  which 
withstand  dessication,  such  as  seeds  or  bacterial  spores,  represent  life 
latent,  but  arrested,  only  to  be  resumed  in  full  vigor  upon  the  readmis- 
sion  of  water. 

The  force  which  impels  the  movement  of  water  into  or  out  of  the 
the  elements  of  living  matter  is  the  difference  between  the  Osmotic 
Pressure  of  the  fluids  within  the  cell  on  the  one  hand,  and  the  external 
medium  which  bathes  the  cell  on  the  other.  The  manner  in  which 
this  force  may  impel  the  migration  of  water  will  be  evident  if  we 
consider  the  mechanism  by  which  it  originates.  We  may  suppose 
that  the  molecules  of  a  substance  in  solution  are  in  a  state  of  con- 
tinuous motion,  as,  indeed,  their  diffusibility  shows  that  they  must 
be.  Let  us  consider  the  condition  of  affairs  in  a  vessel  filled  with 
water  (Fig.  9)  and  divided  into  two  parts  by  a  partition  A-B,  on  the 
right-hand  side  of  which  we  introduce  such  an  amount  of  some  diffus- 
ible substance,  such  as  glucose,  that  there  are  ten  molecules  of  glucose 
in  the  mixture  for  every  ninety  molecules  of  water.  Evidently,  on 
the  left-hand  side  of  it  every  molecule  which  collides  with  this  parti- 
tion will  be  a  water-molecule,  but  on  the  right-hand  side  every  tenth 
molecule  will  be  a  sugar-molecule. 

If,  now,  the  partition  A-B  is  constructed  of  such  material  that  it  is 
porous  to  water,  but  impermeable  for  more  bulky  molecules  such  as 
those  of  sugar,  it  is  evident  that  out  of  100  molecules  bombarding  the 
partition  from  the  left  all  will  pass  through  into  the  right-hand  chamber, 
while  out  of  100  molecules  bombarding  the  partition  from  the  right, 
only  90  will  be  able  to  penetrate  into  the  left-hand  chamber.  In  any 
given  interval  of  time,  therefore,  an  excess  of  water  molecules  will  have 
entered  into  the  right-hand  chamber,  and  this  excess  will  be  directly 
proportionate  to  the  concentration  of  sugar  dissolved  therein. 

Such  a  partition  as  that  which  we  have  described  constitutes  what 
is  known  as  a  Semipermeable  Membrane,  and  membranes  having  the 
characteristic  of  permitting  the  passage  of  water  but  not  of  dissolved 
substances  are  very  numerous.  The  one  most  frequently  employed 


PROPERTIES  CONFERRED  BY  CONSTITUENTS 


257 


for  osmotic-pressure  measurements  is  the  membranous  precipitate  of 
Copper  Ferrocyanide  which  is  formed  when  a  solution  of  copper  sulphate 
comes  into  contact  with  a  solution  of  potassium  ferrocyanide. 

If  the  continuous  entry  of  water  into  the  right-hand  chamber  were 
permitted  and  the  level  of  fluid  did  not  rise  so  as  to  create  a  pressure, 
water  would  pass  indefinitely  from  left  to  right  until  the  sugar  in 
the  right-hand  compartment  was  infinitely  diluted.  In  this  way  no 
measurement  of  the  attraction  of  the  solution  for  water  would  be 
possible,  since,  in  theory,  if  no  frictional  forces  or  pressures  interfered 
with  the  free  motion  of  water,  every  solution,  concentrated  or  dilute, 
would  attract  an  infinite  volume  of  water.  We  may,  however,  measure 
the  degree  of  attraction  for  water  which  is  exerted  by  the  dissolved 
substance  by  determining  the  pressure  or  temperature  necessary  to 


100    H,0 


90   H,0 


10  SUGAR 


B 

FIG.  9 

increase  the  force  or  frequency  of  the  impacts  on  the  right-hand  side 
of  the  partition,  until  the  greater  speed  of  transit  from  right  to  left 
compensates  for  the  greater  volume  of  transit  from  left  to  right.  If 
pressure  be  applied  to  the  contents  of  the  right-hand  compartment, 
the  force  of  the  impacts  of  the  molecules  upon  the  partition  is  increased 
so  that  although  only  ninety  water  molecules  collide  with  the  right- 
hand  side  of  the  partition  for  every  hundred  which  collide  with  the 
left-hand  side,  yet  those  colliding  on  the  right  do  so  more  forcibly, 
and  thus  a  greater  proportion  succeed  in  penetrating  the  membrane, 
until,  when  the  pressure  applied  to  the  solution  attains  a  certain 
magnitude  the  greater  proportion  of  collisions  leading  to  penetration 
of  the  membrane  exactly  balances  the  excess  of  the  total  number  of 
collisions  on  the  side  which  is  bathed  by  pure  water, 
17 


258  THE  PROPERTIES  OF  PROTOPLASM 

The  pressure  which  is  required  to  exactly  compensate  the  greater 
frequency  of  collisions  on  the  side  of  the  membrane  which  is  bathed 
by  pure  water  is  termed  the  Osmotic  Pressure  of  the  solution.  Its 
measurement  may  be  rendered  automatic  by  enclosing  the  solution  in 
a  vessel  to  the  orifice  of  which  a  Manometer  is  attached,  so  that  the 
entry  of  a  very  minute  amount  of  water,  into  the  vessel,  insufficient 
to  appreciably  dilute  the  solution,  causes  a  very  considerable  rise  in 
the  mercury  column  of  the  manometer,  and  a  proportionately  large 
increase  of  pressure.  With  practically  negligible  dilution  of  the 
solution,  therefore,  the  necessary  pressure  is  attained  and  cannot  be 
exceeded,  because  the  os  notic  pressure  having  once  been  attained,  the 
rates  of  entry  and  exit  of  water  into  and  oit  of  the  vessel  become 
equal  and  no  further  changes  of  pressure  or  composition  can  occur. 

Such  a  vessel,  provided  with  a  semipermeable  membrane  and  a 
manometer,  is  termed  an  Osmometer.  Since  the  pressures  which  are 
generated  are  usually  very  great,  the  walls  of  the  vessel  must  be  made 
of  strong  material,  and  the  membrane,  especially,  must  be  constructed 
so  as  lot  to  break  under  the  strain.  These  desiderata  are  attained  by 
employing  a  vessel  composed  of  earthenware,  in  the  minute  pores  of 
which  membranes  are  formed  by  filling  the  vessel  with  a  solution  of 
potassium  ferrocyanide  and  immersing  it  in  a  solution  of  copper 
sulphate.  The  two  reagents  diffuse  outward  and  inward,  or  may  be 
induced  to  do  so  by  electrolysis,  until  they  meet  at  some  point  within 
the  pores  across  which  membranes  of  minute  dia  leter  are  formed. 
Such  membranes  withstand  relatively  enormous  pressures,  while  a 
large  continuous  membrane  would  rupture  under  the  strain  of  much 
smaller  pressures. 

Another  way  of  equalizing  the  rates  of  transposition  of  water  across 
the  membrane  would  be  to  raise  the  Temperature  of  the  solution 
above  that  of  the  water  upon  the  other  side  of  the  membrane.  Increase 
in  temperature  results  in  proportionate  increase  of  the  mobility  of 
the  molecules,  so  that  the  collisions  with  the  membrane  would  be 
proportionately  more  numerous  per  unit  of  time  on  the  heated  than 
on  the  unheated  side.  This  procedure  is  not  practicable  with  the 
membranes  which  we  have  hitherto  been  considering,  because  they 
would  conduct  heat  from  the  one  chamber  to  the  other  and  the  tem- 
peratures of  the  two  compartments  on  either  side  of  the  membrane 
would  soon  be  equalized.  We  may  very  easily  employ  this  method,  how- 
ever, if  in  the  place  of  a  thin  solid  membrane  we  employ  a  layer  of  air. 
Then,  provided  the  dissolved  substance  is  not  volatile,  i.  e.,  soluble  in 
air,  we  have  in  effect  a  semipermeable  membrane  which  is  a  poor  con- 
ductor of  heat  and  which  may  be  obtained  of  any  thickness  which  we  may 
desire.  If  two  chambers  or  vessels,  the  one  containing  water,  the  other 
containing  a  solution,  be  both  placed  in  a  confined  space  or  large  vessel 
filled  with  air,  water  will,  if  the  two  liquids  are  at  the  same  temperature, 
slowly  distil  over  from  the  compart  nent  co  itaining  pure  water  into 
the  compartment  containing  the  solution,  which  thus  becomes  pro- 


PROPERTIES  CONFERRED  BY  CONSTITUENTS  259 

gressively  more  dilute.  If  unchecked  by  any  balancing  or  opposing 
influence,  this  distillation  will  continue  until  the  solution  becomes 
infinitely  dilute,  i.  e.,  practically  equivalent  to  distilled  water.  In 
this  case,  therefore,  as  in  the  case  of  thin  solid  membranes,  we  can 
only  measure  the  attraction  of  the  solution  for  water  by  measuring 
the  change  in  the  condition  of  the  solution  requisite  for  its  neutraliza- 
tion. This  we  may  accomplish  by  heating  the  solution  and  thus 
increasing  the  mobility  of  the  molecules  which  it  contains,  and  so 
increasing  the  number  of  collisions  per  second  of  water-molecules 
with  the  supernatant  layer  of  air.  The  temperature  to  which  we 
must  raise  the  solution  in  order  to  equalize  the  rates  of  distillation 
to  and  from  the  water  and  the  solution  is  proportionate  to  the  increase 
in  the  collisions  per  second  which  is  requisite  to  produce  this  equaliza- 
tion, and  this  in  turn  must  obviously  be  proportionate  to  the  con- 
centration of  the  dissolved  substance.  Thus  if  the  dissolved  sub- 
stance constitutes  one-tenth  of  the  total  molecules  in  the  solution, 
we  must  raise  the  temperature  of  the  solution  sufficiently  to  increase 
the  total  collisions  per  second  by  one-tenth,  in  order  to  render  the 
rate  of  distillation  equal  to  that  of  pure  water  at  the  lower  tempera- 
ture. If  this  rate  of  distillation  is  sufficient  to  cause  ebullition,  i.  e., 
to  render  the  pressure  of  water-vapor  equal  to  that  of  the  atmosphere, 
it  is  evident  that  the  temperature  required  to  attain  it  will  be  higher 
in  the  case  of  the  solution  than  in  the  case  of  pure  water.  Hence, 
the  Boiling-point  of  water  is  raised  by  dissolved  substances,  and  that 
in  proportion  to  their  molecular  concentration. 

There  is  yet  another  way  in  which  we  may  equalize  the  rates  of 
penetration  of  water  from  opposite  sides  of  the  membrane,  and  that 
is  by  cooling  the  water,  and  thus  reducing  the  mobility  of  its  molecules 
relatively  to  those  of  the  solution.  Now  when  a  solution  freezes,  it 
is  not  the  dissolved  substance  that  freezes,  but  the  solvent,  in  this 
case  water.  The  dissolved  substance,  in  fact,  with  certain  intelligible 
exceptions,  crystallizes  out  and  becomes  mechanically  separated  from 
the  solvent  when  the  latter  freezes.  In  such  a  case  the  membrane 
is  furnished  by  the  surface  separating  the  crystals  of  ice  from  the 
remainder  of  the  solution.  If,  now,  reverting  to  the  diagram  in 
Fig.  9,  the  water  in  the  left-hand  compartment  be  sufficiently  cooled, 
relatively  to  the  solution,  water  will  pass  over  from  the  warm  solution 
into  the  cool  chamber  of  pure  water,  because  of  the  greater  mobility 
of  the  molecules  in  the  warm  solution.1  Hence,  in  order  to  accom- 
plish the  withdrawal  of  water  from  the  dissolved  substance  which 
occurs  at  the  freezing-point  the  water  must  be  cooled  to  a  temperature 

1  Ultimately,  however,  the  greater  mobility  of  the  molecules  in  the  solution  will 
fail  to  compensate  for  the  progressively  decreasing  proportion  of  water  molecules  present 
in  the  solution,  and  the  water  compartment  would  have  to  be  further  cooled  in  order 
to  continue  withdrawal  of  water  from  the  solution.  This  is  the  phenomenon  of  "under- 
cooling" which  freezing  salt  solutions  display.  The  correct  freezing-point  is  that  at 
which  the  first  crystal  of  ice  separates,  and  which  is  marked  by  a  sudden  slight  rise  of 
temperature  due  to  the  disengagement  of  the  latent  heat  of  fusion  ol  the  ice. 


260          THE  .PROPERTIES  OF  PROTOPLASM 

below  the  freezing-point  of  pure  water.  Hence,  the  Freezing-point 
of  water  is  lowered  by 'dissolved  substances,  and  that  in  proportion 
to  their  molecular  concentration. 

The  osmotic  pressure  of  a  solution  of  a  diffusible  substance  may 
therefore  be  measured  either  directly,  employing  a  semipermeable 
membrane,  or  indirectly,  by  measuring  the  elevation  of  the  boiling- 
point  or  the  lowering  of  the  freezing-point.  Conversely  the  molecular 
concentration  of  a  dissolved  substance  may  be  estimated  in  the  same 
ways.  The  osmotic  pressure  exerted  by  a  molecular  solution,  that  is, 
by  one  gram-molecule  of  substance  dissolved  in  a  liter  of  water  is 
22.4  atmospheres.  The  elevation  of  the  boiling-point  in  the  same 
solution  is  0.54°,  while  the  depression  of  the  freezing-point  is  1.86°. 
If,  however,  the  dissolved  substance  undergoes  Electrolytic  Dissociation 
then  each  of  the  ions  which  it  yields  exerts  osmotic  pressure  and 
affects  the  boiling-  and  freezing-points  in  the  same  way  as  a  molecule, 
so  that  for  a  substance  completely  dissociated  into  two  ions,  such  as 
sodium  chloride  in  dilute  solution,  the  osmotic  pressure  per  gram- 
molecule  of  dissolved  substance  is  double  the  above-mentioned  figure, 
and  the  molecular  elevation  of  the  boiling-point  and  lowering  of  the 
freezing-point  are  similarly  enhanced.  If  different  solvents  are 
employed  the  osmotic  pressures  obtained  are  the  same  as  those  obtained 
when  water  is  used  as  a  solvent,  provided  the  molecular  condition  of 
the  dissolved  substance  is  the  same  in  both  solvents,  but  if  it  be 
ionized  in  one  and  not  in  the  other,  or  forms  double  molecules  in  one 
and  not  in  the  other  solvent,  the  pressures  observed  will,  of  course, 
differ  from  one  another  in  a  corresponding  manner  and  degree.  The 
magnitude  of  the  effect  upon  the  boiling-  and  freezing-points,  although 
always  proportionate  in  any  one  solvent  to  the  molecular  plus  ionic 
concentration  of  the  dissolved  substance,  differs  with  different  solvents. 

The  osmotic  pressures  of  tissue-fluids  and  of  fluids  expressed  from 
cells  are  usually  estimated  by  the  Cryoscopic  Method  or  measurement 
of  the  lowering  of  the  freezing-point  of  the  solvent,  in  this  case  water. 
This  measurement  is  much  less  tedious  and  less  subject  to  interference 
by  colloidal  admixtures,  etc.,  than  the  direct  measurement  of  pressure 
in  an  osmometer.  The  elevation  of  the  boiling-point  is  usually  not 
applicable  because  of  the  extensive  changes  induced  in  these  solutions 
by  elevated  temperatures,  for  example  the  coagulation  of  proteins 
and  the  transformation  of  bicarbonates  into  carbonates  with  the 
evolution  of  carbon  dioxide.  The  former  of  these  changes  can  be 
obviated,  of  course,  by  measuring  the  elevation  of  the  boiling-point 
under  reduced  pressures  when  ebullition  occurs  at  a  correspondingly 
lower  temperature,  but  the  difficulty  created  by  the  evolution  of 
carbon  dioxide  still  remains. 

The  following  are  illustrative  measurements,  obtained  by  Hamburger 
and  others,  of  the  lowering  of  the  freezing-point  in  blood-sera*  of  various 
species  of  Mammalia: 


PROPERTIES  CONFERRED  BY  CONSTITUENTS          261 

Lowering  of 
Species.  freezing-point. 

Man       .      . 0.526° 

Ox          0.585° 

Horse .      .      0.564° 

Pig 0.615° 

Rabbit .      0.592° 

Dog :...... 0.571° 

Cat.      .  -    fc. .- -.      .      .      .      ^      .      .      .      0.638° 

Sheep 0.619° 

Echidna  hystrix 0.600° 

From  this  table  two  remarkable  facts  are  apparent:  In  the  first 
place  that  the  osmotic  pressure  of  the  blood  of  species  of  mammalia  so 
diverse  as  man,  herbivora,  carnivora  and  the  monotremes  is  extra- 
ordinarily constant,  and  in  the  second  place  that  it  has  the  magnitude 
of  no  less  than  some  eight  atmospheres,  corresponding  to  the  pressure 
exerted  by  a  one-third  molecular  solution  of  a  non-ionized  substance 
such  as  sugar  or  urea,  or  a  one-sixth  molecular  solution  of  sodium 
chloride.  - 

The  osmotic  pressure  of  the  blood-serum,  as  evaluated  from  the 
lowering  of  the  freezing-point,  rises  slightly,  but  unmistakably,  after 
the  absorption  of  the  products  of  digestion  derived  from  the  meal. 
The  Lymph  has  usually  a  higher  osmotic  pressure  than  the  blood,  a 
fact  which  is  attributed  to  the  extraction  of  products  of  metabolic 
activity  from  the  tissues  somewhat  more  rapidly  than  they  can  be 
discharged  from  the  lymph  into  the  blood.  Milk  and  Bile  have  the 
same  osmotic  pressure  as  blood,  Saliva  a  lower  pressure.  Urine  is 
in  general  much  more  concentrated  in  diffusible  constituents  than  the 
blood  or  tissue-fluids  and  therefore  displays  a  much  greater  lowering 
of  the  freezing-point,  usually  between  1.3°  and  2.3°. 

The  blood-sera  of  Birds  possess  an  osmotic  pressure  very  similar  to 
that  of  mammalian  blood -sera.  It  is  a  curious  fact,  however,  that 
the  Eggs  of  birds  have  a  distinctly  lower  osmotic  pressure  than  that 
of  the  blood-serum  of  the  birds  that  lay  them,  or  of  the  blood-serum 
of  the  embryos  that  develop  within  them.  This  is  strikingly  shown 
by  the  following  estimations  of  Atkins. 

Lowering  of  1 
Species.  freezing-point. 

Fowl-blood       .      .- .     0.607° 

Fowl-egg 0.454° 

Duck-blood 0.574° 

Duck-egg 0.452° 

Goose-blood 0.552° 

Goose-egg '.      .      0.420° 

During  Incubation  of  the  egg  the  osmotic  pressure  of  its  contents 
increases  until  it  approximates  to  that  of  the  blood.  Since  in  so  many 
anatomical  particulars  the  Ontogeny  of  the  individual  represents  an 
abbreviated  outline  of  the  Phylogeny  of  the  species,  Atkins  has  sug- 
gested that  the  low  osmotic  pressure  of  the  egg-contents  may  indicate 
descent  of  the  birds  from  ancestors  in  which  the  blood-serum  was  more 
dilute  than  it  is  in  the  birds  of  the  present  epoch.  Since  the  birds 


262  THE  PROPERTIES  OF  PROTOPLASM 

are  probably  descended  from  ancient  forms  of  Reptilia  or  from  forms 
intermediate  between  the  Reptilia  and  the  Amphibia  it  is  of  interest 
to  note  that  many  of  the  Amphibia  and  some  of  the  Reptilia  which 
inhabit  fresh  water  exhibit  a  low  osmotic  pressure  of  the  blood-serum. 
The  following  are  illustrative  figures  cited  after  Hober  and  Jona. 

Lowering  of 

Species.  freezing-point. 

Amphibia: 

Ranaescuknta .      .      .      ,      .      0.465° 

Salamandra  maculata 0.479° 

Reptilia: 

Emys  europaia 0.474° 

Emydura  macquariw 0.550° 

Thalassochelys  caretta 0.610° 

It  will  be  observed  that  the  fresh-water  turtle,  Emys  europaia,  has 
blood  of  which  the  osmotic  pressure  approaches  the  amphibian  type, 
the  marine  turtle,  Thalassochelys  caretta  has  the  avian  and  mammalian 
type  of  osmotic  pressure  of  the  blood,  while  the  tortoise,  Emydura 
macquarice  represents  an  intermediate  pressure.  The  osmotic  pressure 
of  amphibian  blood-serum  closely  approaches  in  magnitude  the  pres- 
sures obtaining  in  the  eggs  of  birds. 

Among  the  various  orders  of  Fishes  in  the  Teleostomi  or  bony 
fishes,  which  are  phylogenetically  the  most  recent  and  highly  developed 
forms,  the  osmotic  pressure  of  the  blood-serum  approximates  much 
more  nearly  to  that  of  the  blood  of  Mammalia,  Aves  and  Reptilia 
than  to  the  osmotic  pressure  of  the  ocean  which  the  marine  forms 
inhabit.  In  the  phylogenetically  older  and  less  specialized  forms,  the 
Elasmobranchii  or  sharks,  however,  the  osmotic  pressure  of  the  blood 
approximates  that  of  sea-water,  as  the  following  figures,  cited  after 
Bottazzi,  reveal: 

Lowering  of 

Fluid.  freezing-point. 

Sea-water 2.30° 

Elasmobranchii : 

Torpedo  marmorata .-     .  2.26° 

Mustelus  vulgaris   .      .      .      . *    .      .-.    .      .  2.36° 

Trygon  violacea 2.44° 

Marine  teleostomi : 

Charax  puntazzo 1.04° 

Cernagigas       . 1.04° 

Crenilabrus  pavo 0.75° 

Box  salpa 0.84° 

Fresh- water  teleostomi: 

Anguilla  vulgaris 0.58°-0.69° 

Barbus  fluviatilis 0.475°-0.558° 

Lcuciscus  dobula 0.45° 

Perca  fluviatilis     1 

Cyprinus  carpio    [  n  _   0 

Tinea  vulgaris       ( 

Esox  lucius 

In  lower  marine  forms  the  tissue-fluids  approximate  still  more 
closely  in  composition  and  concentration  to  the  sea-water  which  these 
organisms  inhabit.  The  following  are  results  obtained  by  Bottazzi: 


OSMOTIC  PRESSURE  OF  CELL  CONTENTS  263 

Lowering  of 
freezing-point  of 

Organism.  tissue-fluids. 
Ccelenterata : 

Alcyonium  palmatum 2.196° 

Echinodermata : 

Asteropecten  aurantiacus 2.312° 

Holothuria  tubulosa 2.315° 

Vermes: 

Sipunculus  nudus         2.31° 

Crustacea: 

Majasquinado 2.36° 

H omarus  vulgaris 2.29° 

Cephalopoda : 

Octopus  macropus 2.24° 

With  the  enhanced  specialization,  therefore,  which  characterizes 
the  higher  and  especially  the  vertebrate  forms  of  life,  independence 
of  the  external  milieu  has  been  acquired  and  the  cells  are  bathed 
in  a  medium  of  relatively  constant  concentration  and,  as  we  shall 
see,  of  even  more  constant  composition. 


THE  OSMOTIC  PRESSURE  OF  CELL-CONTENTS. 

The  osmotic  pressure  of  cell-contents  can,  of  course,  be  determined 
indirectly  by  expressing  the  cell-sap  and  determining  its  freezing- 
point.  In  many  cases,  however,  the  measurement  may  be  made  in 
a  very  much  more  convenient  manner  by  employing  the  method  of 
Plasmolysis  devised  in  1884  by  the  Dutch  botanist,  de  Vries. 

In  many  plants  the  protoplasm  of  the  cells  lies  closely  adherent  to 
the  cellulose  cell-wall,  and  it  is  found  if  these  cells  be  immersed  in 
concentrated  solutions  of  salts,  sugars,  urea  or  other  diffusible  sub- 
stances, that  the  protoplasm  shrinks  away  from  the  supporting  wall 
of  cellulose,  indicating  that  the  protoplasm  has  diminished  in  volume. 
This  loss  of  volume  can  only  be  due  to  the  abstraction  of  water  from 
the  protoplasm,  and  since  the  agencies  which  accomplish  this  abstrac- 
tion of  water  are  solutions  of  relatively  high  osmotic  pressures,  we 
infer  that  the  external  limiting  membranes  of  the  cells,  within  the 
cellulose  cell-wall  but  bounding  the  exterior  of  the  protoplasmic  con- 
tents, is  Semipermeable,  admitting  water  but  not  admitting  a  variety 
of  diffusible  dissolved  substances. 

If  this  interpretation  be  the  correct  one,  then  any  solution  having 
a  higher  osmotic  pressure  than  the  cell-fluids  will  cause  plasmolysis, 
while  the  solutions  which  are  of  just  the  same  osmotic  pressure  as 
the  cellular  fluid  will  fail  to  cause  plasmolysis.  The  solutions  which 
just  fail  to  cause  plasmolysis,  or  which  are  Isotonic  with  the  cell- 
fluids,  should  therefore  all  be  of  the  same  molecular  or  molecular 
plus  ionic  concentration,  independently  of  the  nature  of  the  dissolved 
substance,  provided,  only,  that  it  is  not  able  to  penetrate  the  cell- 
membrane  in  measurable  proportion  within  the  period  of  time  con- 
sumed by  the  shrinkage  of  the  protoplasm. 


264  THE  PROPERTIES  OF  PROTOPLASM 

The  following  are  results  which  were  obtained  by  Over  ton,  employ- 
ins;  the  cells  of  spirogyra  filaments: 

Isotonic  concentration: 

Dissolved                                                       Molecular                     found,  calculated, 

substance.                                                         weight.  per  cent.  per  cent. 

Cane-sugar .      .     342  6.0 

Mannitol 182                         3.5  3.19 

Glucose 180                        3.3  3.15 

Arabinose 150                         2.7  2.63 

Erythritol 122  2.14 

Asparagin 132  2.5 

GlycocoU 75                          1.3  1.32 

The  tf calculated"  values  were  computed  as  follows:  The  isotonic 
concentration  of  cane-sugar  being  6  per  cent,  and  its  molecular  weight 
342,  the  concentration  of  an  isotonic  solution  is  evidently  /£$  =  ^ 
molecular.  A  ^y  molecular  solution  of  glycocoll  would  contain  |^- 
grams  of  glycocoll  per  liter,  or  1.32  grams  per  hundred  c.c.  It  will 
be  seen  that.the  experimental  and  the  calculated  values  are  exceedingly 
close  to  one  another  and  we  may  infer  that,  at  all  events  so  far  as 
limited  periods  of  time  are  concerned,  the  protoplasm  of  spirogyra  is 
impermeable  to  the  substances  mentioned,  although  freely  permeable 
to  water. 

This  method  of  estimating  the  isotonicity  of  solutions,  however,  is 
subject  to  several  sources  of  error  and  uncertainty.  In  the  first  place 
we  must  take  into  consideration  the  fact  that  the  protoplasmic  limit- 
ing membrane  must  necessarily  alter  in  form  before  we  can  perceive 
any  solution  to  be  Hypertonic,  or  in  excess  of  the  isotonic  concentra- 
tion. Now  the  external  limiting  membranes  of  cells  must  undoubtedly 
possess  some  degree  of  Elasticity,  in  consequence  of  which  they  must 
themselves  exert  some  pressure  upon  the  cell-contents.  The  forces 
leading  to  shrinkage  of  the  protoplasm  are  not  solely  osmotic  there- 
fore, but  to  some  slight  extent  elastic  also,  and  we  cannot  positively 
estimate  the  proportion  of  the  total  force  which  this  elasticity  com- 
municates, since  it  will  not  improbably  add  a  constant  amount  to 
each  osmotic  pressure  investigated.  Isotonic  solutions  are  therefore 
isosmotic  with  one  another,  but  not  necessarily  isosmotic  with  the 
cell-contents.  In  red  blood-corpuscles  this  is  probably  the  origin  of 
the  constant  slight  difference  between  the  osmotic  concentration  of  the 
contents  of  the  corpuscles  and  the  surrounding  medium  or  plasma, 
amounting,  according  to  Moore  and  Roaf,  to  a  difference  of  freezing- 
point  depression  of  0.02°  to  0.03°  C.,  or  an  osmotic-pressure  difference 
of  from  0.24  to  0.36  of  an  atmosphere.  -t 

In  the  second  place,  the  Semipermeability  of  living  cell-membranes 
is,  of  necessity,  never  absolute.  This  becomes  obvious  when  we 
consider  that  the  nutrition,  and  therefore,  the  maintenance  and 
growth  of  cells  depends  upon  their  intake  of  substances  dissolved 
in  water.  Unless  a  cell  can  be  penetrated  by  the  mineral  or  organic 
substances  which  constitute  the  components  out  of  which  protoplasm 
is  built  up,  the  progressive  consumption  of  material  and  dissipation 


OSMOTIC  PRESSURE  OF  CELL-CONTENTS  265 

of  energy  by  the  cell  must  rapidly  lead  to  its  disintegration.  Further- 
more, the  solutions  which  Overton,  in  the  results  cited  above,  found 
to  be  isotonic  with  the  cell-contents  of  spirogyra  filaments  exerted 
an  osmotic  pressure  of  some  four  and  a  half  atmospheres,  and  the 
corresponding  pressure  in  the  cell-contents  themselves,  can  only 
have  been  due  to  diffusible  water-soluble  substances  which  must 
therefore  have  penetrated  the  protoplasm  at  some  period  of  its  devel- 
opment. The  semipermeability  of  cell-membranes  is  in  fact,  even 
in  the  most  typical  instances,  apparent  and  not  real.  It.  is  purely  a 
question  of  Relative  Permeability,  of  the  rapidity  with  which  dissolved 
substances  and  water  relatively  penetrate  the  cell.  In  the  case  of 
Bacillus  cholera,  for  example,  the  relativity  of  the  semipermeability 
of  cells  can  very  clearly  be  seen,  for  these  organisms,  as  well  as  certain 
other  bacteria,  are  temporarily  plasmolyzed  by  hypertonic  salt  solu- 
tions or  sugar-solutions,  but  not  at  all  by  Glycerol  solutions.  Even 
the  plasmolysis  observed  in  salt-  or  sugar-solutions  disappears  in  the 
course  of  an  hour  or  two,  because,  after  the  lapse  of  this  time,  a  suffi- 
cient proportion  of  the  salt  or  sugar  has  penetrated  the  cells  to  restore 
isotonicity  between  the  inner  and  outer  fluids.  Evidently,  therefore, 
in  the  case  of  these  cells  water  and  glycerol  penetrate  the  exterior 
limiting  membrane  almost  instantaneously,  salt  and  sugar  more 
slowly.  The  disparity  of  the  velocities  of  penetration  for  water  and 
dissolved  substances  is  greater  in  spirogyra  filaments  than  in  the 
above-mentioned  species  of  bacteria,  and  this  constitutes  the  origin 
of  the  apparent  semipermeability  of  the  protoplasm  in  spirogyra. 

It  is  a  rather  remarkable,  and  certainly  a  regrettable  fact  that 
physical  chemists  have  hitherto  paid  so  little  attention  to  the  investi- 
gation of  the  Time-relation  of  Osmosis.  The  comparative  neglect  of 
this  and  other  fields  of  inquiry  which  would  naturally  suggest  them- 
selves to  the  student  of  pure  physics  or  mechanics,  is  undoubtedly 
attributable  to  the  bias  toward  purely  thermodynamical  reasoning 
which  has  been  communicated  to  the  students  of  physical  chemistry 
by  the  past  generation  of  chemists.  The  thermodynamical  relation- 
ships and  equations  contemplate  only  attained  equilibria,  not  fluctuat- 
ing or  kinetic  phenomena.  Hence,  the  relationship  between  the  lapse 
of  time  and  the  degree  of  penetration  of  a  'membrane  by  various 
solvents  or  dissolved  substances,  which  would  seem  to  present  a  most 
obvious  subject  for  inquiry,  is  as  yet  very  imperfectly  known.  One 
would  expect,  however,  that  the  quantity  of  penetration  would  be  an 
exponential  function  of  the  time,  and  that  this  function  would  contain 
specific  parameters  or  constants,  characteristic  for  the  membrane, 
the  particular  solvent  employed,  and  the  dissolved  substance  respec- 
tively. The  evaluation  of  these  parameters  in  the  case  of  living  cell- 
membranes  would  afford  an  accurate  quantitative  measure  of  Per- 
meability, for  the  estimation  of  which  we  must  rely  at  present  upon 
qualitative  rather  than  upon  quantitative  data. 

In  the  plasmolytic  method  of  estimating  isotonic  solutions  we  regard 


266  THE  PROPERTIES  OF  PROTOPLASM 

as  isotonic  those  solutions  which  are  just  insufficiently  Hypertonic 
to  cause  withdrawal  of  water  from  the  cell-contents.  The  effects  of 
Hypotonic  solutions  or  solutions  which  are  more  dilute  than  the  cell- 
content  are  more  readily  studied  in  cells  which  possess  no  rigid  support- 
ing framework,  such  as  the  exterior  cellulose  wall  of  plant-cells.  The 
Red  Blood-corpuscles  were  first  employed  by  Hamburger  for  this 
purpose.  If  these  cells  are  suspended  in  sufficiently  hypotonic  solu- 
tions, the  excessive  penetration  of  water  into  the  cells  results  in  their 
rupture  by  the  internal  pressure  which  results,  and  hemoglobin  is 
set  free,  tingeing  the  supernatant  fluid  red.  The  technique,  therefore, 
consists  in  suspending  the  corpuscles  in  solutions  of  varying  concen- 
tr'ation  and  allowing  them  to  settle  to  the  bottom  of  the  tube.  A 
solution  which  is  just  sufficiently  hypotonic  to  burst  some  of  the 
corpuscles  will  be  tinged  with  hemoglobin  and  the  corpuscles  are  then 
said  to  have  undergone  Hemolysis. 

The  degree  of  hypotonicity  required  to  rupture  red  blood-corpuscles 
is  apparently  the  same  for  a  variety  of  dissolved  substances,  so  that 
the  solutions  are  found  to  be  isotonic  with  one  another,  as  the  following 
data  show: 

Limiting  concentration 

which  causes 
Substance.  Molecular  weight.  hemolysis,  per  cent. 

NaCl ,58.5  0.585 

CHsCOOK     .....;....     98.1  1.04 

KNO3  .      .      .      .......      .      .101.1  1.00 

NaBr  .      .      .      ...     .     , 102.9  1.02 

Nal 149.9  1.55 

KI        .      .      .      .      .      .     .      ....      .    166.0  1.65 

Solutions  which  cause  hemolysis,  although  isotonic  with  one  another 
are,  of  course,  by  no  means  isotonic  with  the  fluid  contents  of  the 
corpuscles,  for  the  bursting  of  the  cells  indicates  not  a  slight  but  a  very 
considerable  excess  of  pressure  within  them.  Solutions  insufficiently 
hypotonic  to  cause  actual  rupture  of  the  cells  will  nevertheless  cause 
them  to  swell  through  absorption  of  water,  while  slightly  hypertonic 
solutions  will,  on  the  contrary,  cause  shrinkage  of  the  cells  through  the 
withdrawal  of  water,  just  as  in  the  plasmolysis  of  plant-cells.  This  is 
the  foundation  of  the  Hematocrit  method  of  measuring  isotonicity, 
devised  by  Hedin  and  Koeppe.  Blood-corpuscles,  freed  from  serum 
by  washing  them  with  isotonic  salt  solution,  are  suspended  in  measured 
amounts  of  various  solutions  to  be  investigated,  and  the  mixtures  are 
placed  in  specially  constructed  centrifuge-tubes  of  very  narrow  bore 
and  provided  with  fine  graduations.  The  tubes  are  then  centrifuged 
and  the  heights  of  the  columns  of  corpuscles  compared  in  the  various 
tubes.  If  the  corpuscles  have  swollen  they  will  occupy  a  larger  volume 
in  the  tube,  if  they  have  lost  water  they  will  occupy  a  smaller  volume 
than  the  corpuscles  which  are  immersed  in  strictly  isotonic  salt  solu- 
tions. From  the  lowering  of  the  freezing-point  we  know  that  blood- 
serum  is  isotonic  with  ™  NaCl  or  f  sugar  solutions,  and  it  is  experi- 
mentally found  that  in  the  majority  of  instances  salt  solutions  which 


OSMOTIC  PRESSURE  OF  CELL  CONTENTS  267 

slightly  exceed  this  concentration  cause  shrinkage  of  the  corpuscles, 
while  solutions  which  are  less  concentrated  than  blood-serum  cause 
swelling  of  the  corpuscles. 

Assuming  the  corpuscles  in  normal  serum  to  be  withstanding  no 
pressure  or,  at  the  most,  a  very  slight  one,  it  is  of  some  interest  to 
calculate  from  the  degree  of  hypotonicity  the  pressure  which  is  required 
to  rupture  the  corpuscles  so  as  to  discharge  hemoglobin  into  the  solu- 
tion. The  solutions  which  are  just  sufficiently  concentrated  to  prevent 
rupture  are,  as  we  have  seen,  isotonic  with  a  one-tenth  molecular 
sodium  chloride  solution  or,  which  comes  in  terms  of  osmotic  pressure 
to  the  same  thing,  a  one-fifth  molecular  solution  of  sugar.  When 
neither  swollen  nor  shrunken  these  cells  are  isotonic  with  a  one  third 
molecular  solution  of  sugar.  The  degree  of  hypotonicity  required  to 
rupture  the  cells  therefore,  corresponds  to  the  pressure  exerted  by  a 
3  —  i  =  A  molecular  solution  of  sugar,  i.  e.,  to  a  pressure  of  about 
three  atmospheres. 

From  the  data  which  we  have  cited  the  surface  of  a  red  blood-cor- 
puscle would  appear  to  afford  an  example  of  a  strictly  semipermeable 
membrane.  Here  again,  however,  semipermeability  is  relative  and 
not  absolute.  Not  only  water  can  enter  the  cells  with  ease  but  also 
other  substances  with  varying  difficulty.  An  ingenious  method  of 
illustrating  this  fact  is  that  which  has  been  devised  by  Hedin. 

A  measured  amount  of  the  substance  for  which  the  permeability  of 
the  corpuscles  is  to  be  tested  is  dissolved  in  defibrinated  blood,  i.  e., 
in  a  mixture  of  serum  and  corpuscles.  The  serum  of  this  blood  will 
be  found  to  freeze  at  a  lower  temperature  than  untreated  serum, 
because  a  certain  proportion  of  an  additional  diffusible  substance  is 
contained  in  it.  The  depression  of  the  freezing-point  of  this  serum 
may  be  designated  "a."  Now  to  an  equal  volume  of  serum  which 
does  not  contain  any  corpuscles  an  equal  amount  of  the  same  substance 
is  added.  This  serum  will  also  freeze  at  a  lower  temperature  than 
normal  serum,  and  the  depression  of  the  freezing-point  which  it  exhibits 
may  be  designated  "b."  Now,  it  is  evident  that  if  the  substance 
which  was  added  to  the  defibrinated  blood  penetrated  the  corpuscles 
and  dissolved  in  them  to  the  same  extent  as  in  an  equal  volume  of 
serum,  the  concentrations  of  the  substance  in  the  two  samples  of 
serum  would  be  equal  to  one  another,  and  we  would  have  a  =  b.  If  the 
blood  corpuscles  in  the  defibrinated  blood  took  up  less  of  the  dissolved 
substance  than  an  equal  volume  of  serum,  then  the  substance  would 
be  present  in  greater  concentration  in  the  first  sample  of  serum  than  in 
the  second,  and  we  would  have  a  >  b  or  jj  >  1.  If,  on  the  other  hand, 
the  blood-corpuscles  took  up  more  of  the  dissolved  substance  than  an 
equal  volume  of  serum  then  we  would  have  a  <  b  or  ^  <  1 . 

The  results  of  this  method  show  that  the  salts  of  the  alkalies  and 
alkaline  earths  and  the  amino-acids  and  sugars  penetrate  the  corpuscles 
with  great  difficulty.  Ammonium  Salts  and  Urea,  however,  pass  into  the 


268  THE  PROPERTIES  OF  PROTOPLASM 

corpuscles  readily.  Among  the  alcohols  there  is  an  interesting  relation- 
ship between  the  number  of  hydroxyl-groups  which  they  c6ntain  and 
the  readiness  with  which  they  penetrate  the  corpuscles.  The  hexa- 
tomic  and  pentatomic  alcohols  hardly  penetrate  the  corpuscles  at  all. 
Erythritol,  which  is  a  tetra-atomic  alcohol  arid  Glycerol  which  is  tria- 
tomic  penetrate  slowly.  Ethylene  Glycol,  which  is  a  diatomic  alcohol, 
penetrates  the  cells  rather  rapidly,  while  the  Monatomic  Alchols  divide 
themselves  immediately  in  equal  proportion  between  the  corpuscles 
and  the  serum.  Ether,  esters,  aldehyde  and  acetone,  on  the  other 
hand,  are  preferentially  absorbed  by  the  corpuscles,  so  that  they 
become  more  concentrated  in  the  corpuscles  than  in  the  serum  which 
bathes  them.  Of  course  only  those  substances  which  fail  to  enter  the 
cells  quickly  can  cause  shrinkage  of  the  corpuscles  in  hypertonic 
solutions. 


THE  COMPOSITION  OF  THE  MINERAL  CONSTITUENTS  OF 
TISSUE  FLUIDS. 

It  was  first  pointed  out  by  Ringer  in  1882  that  the  relative  propor- 
tions of  the  mineral  constituents  in  the  blood-sera  of  different  mam- 
mals are  most  remarkably  constant  and,  furthermore,  that  notwith- 
standing the  fact  that  potassium  and  calcium  salts  are  present  in  blood- 
serum  only  in  minute  proportion  relatively  to  the  sodium  salts,  yet 
their  presence  in  the  established  proportion  is  actually  essential  to  the 
proper  performance  of  their  functions  by  the  tissues,  a  very  slight 
alteration  in  the  mineral  composition  of  the  fluid  bathing  them  being 
very  deleterious. 

On  the  basis  of  numerous  analyses  of  the  ash  of  blood-sera,  the 
following  .composition  was  established  by  Locke  as  the  most  suitable 
circulating  fluid  for  mammalian  tissues:  NaCl,  0.9  per  cent.;  KC1, 
0.042  per  cent.;  CaCl2,  0.024  per  cent.  To  this  mixture  a  small  propor- 
tion (0.01  to  0.03  per  cent.)  of  sodium  bicarbonate  is  generally  added  to 
neutralize  the  acids  which  are  produced  by  tissue-activities  and  a  little 
glucose  (0.1  per  cent.)  has  been  shown  to  prolong  the  life  of  excised 
tissues  which  are  kept  for  prolonged  periods  in  this  artificial  circulating 
fluid.  The  glucose  is  consumed  by  the  tissue  and  probably  serves  as  a 
nutrient.  When  the  glucose  is  omitted  this  mixture  is  usually  desig- 
nated Ringer's  Solution. 

For  amphibian  tissues  a  slightly  more  dilute  solution  is  employed. 
The  solution  originally  recommended  by  Ringer  was  a  0.6  per  cent, 
solution  of  sodium  chloride  saturated  with  calcium  phosphate  to  which 
0.03  per  cent,  of  potassium  chloride  was  added.  A  suitable  fluid  may 
also  be  prepared  by  simply  adding  to  Locke's  Solution  one-third  of  its 
volume  of  distilled  water.  Loeb  has  pointed  out  that  the  proportions 
of  the  various  salts  in  Ringer's  and  Locke's  solutions  correspond 
approximately  to  the  ratios:  100  molecules  of  NaCl  to  2  molecules 


COMPOSITION  OF  THE  MINERAL  CONSTITUENTS          269 

of  KC1  to  2  molecules  of  CaCl2,  the  total  concentration  being  one-sixth 
molecular.' 

Not  only,  however,  are  the  mineral  constituents  of  mammalian 
serum  constant  in  composition,  but  even  in  the  blood  of  fishes  we  find 
that  substantially  the  same  relative  proportions  obtain.  The  following 
analyses  are  cited  after  Macallum,  the  percentage-concentTSition  of 
sodium  being  taken  as  100  and  the  percentages  of  the  remaining  metals 
reduced  to  the  same  units: 

Species.  Na  K  Ca  Mg 

Dogfish  (A canthias  vulgaris)        .100  4.6  2.7  2.5 

Cod  (Gadus  callarias)        ...  100  9.5  3.9  1.4 

Pollock  (Pollachius  mrens)      .      .  100  4.3  3.1  1.5 

Dog 100  6.9  2.5  0.8 

Mammal  (average)      ....  100  6.7  2.6  0.8 

Man 100  6.1  2.7  0.9 

The  remarkable  uniformity  of  composition  which  is  thus  displayed 
by  the  blood-sera  of  such  diverse  organisms,  suggests  that  it  is  deter- 
mined by  some  common  cause,  more  especially  since  a  slight  alteration 
of  the  normal  mineral  composition  of  blood-sera  causes  profound 
disturbance  of  the  functions  of  the  tissues.  The  interesting  suggestion 
has  been  put  forward  by  Macallum  that  the  mineral  composition  of 
vertebrate  blood-sera  represents  the  composition  of  the  sea-water  at  the 
time  when  the  early  ancestors  of  the  present  vertebrate  forms  first 
acquired  an  organ,  namely  the  kidney,  of  which  the  function  is  to 
maintain  constancy  of  composition  in  the  body-fluids.  In  the  lower 
marine  forms  of  the  present  day  which  do  not  possess  any  corresponding 
excretory  organ,  the  composition  and  concentration  of  the  body-fluids 
is  practically  identical  with  that  of  the  sea-water  in  which  they  live, 
but  in  mammals  the  tissue-fluids  are  not  only  more  dilute  than  present- 
day  sea-water,  but  they  differ  from  it  in  containing  a  much  smaller 
proportion  of  one  mineral  constituent,  namely  Magnesium.  The  fol- 
lowing figures  are  cited  after  Macallum,  the  percental-concentration 
of  sodium,  as  before,  being  taken  as  100  and  the  percentages  of  the 
remaining  metals  reduced  to  the  same  standard. 

Fluid.  Na  K  Ca  Mg 

Ocean-water 100  3.6  3.9  12.1 

Tissue-fluid  of  a  jellyfish  (Aurelia 

flavidula) 100  5.2  4.1  11.4 

Blood-serum  of  a  dog        ...  100  6.9  2.5  0.8 

The  correspondence  of  the  three  sets  of  figures,  excepting  in  regard  to 
magnesium,  is  certainly  striking  and  the  oceanic  origin  of  these  widely- 
found  ratios  appears  very  probable. 

Among  the  crustaceans  the  more  primitive  forms,  such  as  Limulus 
possess  a  blood-serum  which  is  practically  of  the  same  composition  as 
sea-water.  In  more  highly  developed  forms  such  as  Homarus  an 

1  The  actual  ratios  in  Locke's  solution  are  100  NaCl  :  3.6  KC1  :  1.4  CaCl?,  In  sea- 
water  the  ratios  are:  100  NaCl  :  2.2  KCJ  :  1.5  CaCl?, 


270  THE  PROPERTIES  OF  PROTOPLASM 

approach  toward  the  vertebrate  composition  of  the  serum  is  already 
indicated,  as  the  following  figures  reveal: 

Fluid.                                                  Na  K  Ca  Mg 

Ocean-water 100  3.6  3.9  12.1 

Serum  of  Limulus  polyphemus    .100  5.6  4.1  11.2 

Serum  of  Homarus  americanus    .100  3.7  4.9  1.7 

According  to  Macallum  the  development  of  a  kidney  in  the  proto- 
vertebrate  forms  from  which  vertebrates  have  arisen,  fixed  the  com- 
position of  the  tissue-fluids  of  the  vertebrata  for  all  time,  since  the 
primitive  kidney  was  adapted  to  the  concentration  and  proportions  of 
the  mineral  constituents  of  the  ocean  of  that  period.  In  the  early 
Cambrian  or  pre-Cambrian  period  at  which  the  ancestral  forms  of  the 
vertebrates  arose,  the  sea-water  must  have  been  very  much  more  dilute 
than  it  is  at  present  day,  because  sodium  chloride  is  constantly  accumu- 
lating, since  it  is  not  deposited  in  important  amounts  in  the  marine 
geological  formations.  Calcium  and  potassium  are  deposited  from  sea- 
water  in  the  form  of  limestone  and  minerals  such  as  glauconite  at  about 
the  same  rate  as  that  at  which  they  are  carried  into  the  sea  by  rivers. 
Magnesium,  however,  is  increasing  in  the  sea-water  not  only  absolutely 
but  also  relatively  to  the  sodium,  the  rate  of  deposition  being  much 
slower  than  the  rate  of  addition.  It  is  quite  probable,  therefore,  that 
the  sea-water  of  the  early  Cambrian  epoch  was  not  only  much  more 
dilute  than  the  sea-water  of  our  day,  but  also  contained  both  absolutely 
and  relatively  much  less  magnesium. 

The  blood-serum  of  mammals  therefore  resembles  a  diluted  sea- 
water  with  the  exception  that  its  magnesium  content  is  both  absolutely 
and  relatively  much  lower  than  the  magnesium  content  of  the  sea-water 
of  our  own  day.  Just  as  the  homoiothermal  animals  have  acquired  a 
large  measure  of  independence  of  the  temperature  of  their  environment, 
so,  and  at  an  earlier  stage  of  evolution,  the  vertebrates  have  acquired 
a  large  measure  of  independence  of  their  osmotic  environment, — they 
are  "homoiosmotic,"  while  the  more  elementary  forms  are  "poikilos- 
motic"  and  the  cells  of  which  they  are  composed  are  exposed  to  all  the 
disadvantages  of  an  irregularly  fluctuating  milieu.  At  a  still  earlier 
stage  of  evolution  the  multicellular  organisms  acquired,  as  we  shall  see, 
more  or  less  efficient  means  of  maintaining  constancy  of  the  reaction 
or  hydrogen  ion  concentration  of  their  tissue-fluids.  Each  of  these 
successive  stages  marked  an  additional  degree  of  emancipation  from 
the  fortuitous  inequalities  of  an  unstable  environment  and  a  step 
toward  the  self-creation  of  an  equable  "internal  environment/'  suit- 
able for  the  maximum  furtherance  of  vital  activities. 

The  mechanism  by  which  this  environmental  stability  is  brought 
about  is  similar  in  each  of  the  three  cases  and  consists  in  a  balance 
between  income  and  output  so  adjusted  that  the  dissipating  agencies 
(excretory  activity  of  the  kidneys,  radiation  of  heat  from  the  surface 
of  the  body,  and  release  of  carbon  dioxide  from  the  lungs,  respectively) 


NEUTRALITY  OF  THE  TISSUES  AND  TISSUE-FLUIDS        271 

discharge  their  functions  under  the  stimulus  of  a  definite  positive  or 
negative  pressure,  acting  like  so  many  dams,  to  maintain  the  reservoir 
of  mineral  constituents,  heat,  or  bases  at  a  certain  height  while  the 
inflow  and  outflow  are  equalized  so  that  the  height  of  the  reservoir 
does  not  progressively  increase  or  decrease.  We  have  in  fact  in  each 
case  a  number  of  balanced  activities  in  dynamic  equilibrium,  a  type  of 
mechanism  which  is  repeatedly  reduplicated  in  life-phenomena. 

Notwithstanding  the  fact  that  the  mineral  composition  of  mam- 
malian blood-sera  differs  appreciably  from  that  of  sea-water  only  in 
total  concentration  and  in  the  relative  content  of  a  single  constituent, 
Magnesium,  yet  this  latter  difference  renders  sea-water,  even  when 
diluted  to  isotonicity  with  blood-serum,  far  from  a  physiologically 
neutral  fluid  for  mammalian  tissues.  It  has  been  shown  by  Burnett 
that  sterilized  sea-water,  rendered  isotonic  with  blood-serum  by 
dilution,  causes  Glycosuria,  considerable  amounts  of  glucose  appearing 
in  the  urine  when  the  sea-water  is  injected  into  the  circulation  of 
rabbits.  The  same  effect  is  brought  about  by  Locke's  or  Ringer's 
solutions,  if  magnesium  is  added  to  them  in  the  proportion  in  which 
it  is  present  in  sea-water.  Hence  diluted  and  sterilized  sea-water 
cannot  be  employed  for  surgical  purposes  as  a  substitute  for  Locke's 
or  Ringer's  solution. 

THE  NEUTRALITY  OF  THE  TISSUES  AND  TISSUE-FLUIDS. 

The  statements  concerning  the  alkalinity  of  the  blood  which  are  to  be 
found  in  the  physiological  and  medical  literature  of  the  last  and  early 
part  of  this  century  are  totally  unreliable  since  they  were  based  upon 
the  erroneous  belief  that  it  is  possible  to  ascertain  the  reaction  of  such 
a  fluid  as  the  blood  by  titration.  The  method  of  titration  merely 
informs  us  of  the  quantity  of  bases  which  are  present  either  uncombined 
or  else  combined  with  weak  acids  such  as  carbonic  acid,  which  are  dis- 
placed from  their  compounds  by  the  stronger  acids  used  in  titration. 
If  all  of  the  bases  are  present  in  the  free,  uncombined  form  then,  in 
dilute  solutions  at  all  events,  the  true  alkalinity  or  hydroxyl  ion  con- 
centration may  be  fairly  accurately  estimated  to  be  equivalent  to  the 
amount  of  acid  required  for  neutralization.  But  this  is  not  at  all  the 
case  if.  the  bases  are  partially  or  wholly  combined  with  weak  acids, 
because  in  that  event  the  addition  of  the  acid  employed  in  titration 
displaces  the  weak  acid  which,  when  uncombined,  by  reason  of  its 
slight  dissociability,  ceases  to  affect  materially  the  reaction  of  the 
fluid,  and  the  condition  which  we  are  seeking  to  measure  alters  con- 
tinuously throughout  the  titration.  Thus  it  is  possible  to  estimate  all 
of  the  sodium  in  a  solution  of  sodium  bicarbonate  by  direct  titration 
with  sulphuric  acid,  using  Methyl  Orange  as  an  indicator,  because  the 
carbon  dioxide  which  is  displaced  by  the  sulphuric  acid,  is  so  slightly 
dissociated  in  comparison  with  the  acid  used  for  titration  that  its  con- 
tribution to  the  final  reaction  of  the  mixture  is  negligible.  Yet  a 


272 


THE  PROPERTIES  OF  PROTOPLASM 


solution  of  sodium  bicarbonate  is  far  from  possessing  the  alkalinity 
of  a  solution  of  free  sodium  hydroxide  of  the  same  concentration, 
although  so  far  as  the  results  of  titratiori  reveal  there  is  no  distinction 
between  them. 


The  blood  and  other  tissue-fluids  contain  a  large  proportion  of  the 
sodium  salts  of  weak  acids,  namely  carbonic  acid,  phosphoric  acid  and 
proteins.  When  blood-serum  is  titrated  with  hydrochloric  or  sulphuric 


NEUTRALITY  OF  THE  TISSUES  AND  TISSUE-FLUIDS         273 

acid,  using  methyl  orange  as  an  indicator,  by  the  time  the  red  color 
appears  these  compounds  have  been  successively  decomposed,  and,  in 
fact,  some  proportion  of  the  acid  employed  in  the  titration  has  actually 
entered  into  combination  with  the  proteins  which  are  now  acting  as 
bases  instead  of  acting,  as  they  do  in  normal  blood,  as  weak  acids. 
The  Titratable  Alkalinity  of  the  blood,  therefore,  bears  no  relationship 
to  its  actual  alkalinity  or  Hydroxyl  ion  Concentration.  It  does,  how- 
ever, bear  some  relationship,  as  we  shall  see  to  the  power  of  the  blood 
to  maintain  its  neutrality,  in  other  words  to  the  "Alkali-reserve"  of 
blood. 


FIG.  11. — Modified  Cottrell  hydrogen  electrode.     (After  Schmidt.) 


In  order  to  ascertain  the  actual  reaction  of  a  complex  mixture  of 
weak  and  strong  acids  and  bases  such  as  the  blood  or  other  tissue- 
fluids,  therefore,  a  method  of  measurement  must  be  employed  which  is 
static  and  not  dynamic,  i.e.,  which  leaves  the  state  of  the*  blood  unal- 
tered in  respect  to  the  balance  of  acids  and  bases  which  it  contains. 
For  this  purpose  no  method  is  better  adapted  for  obtaining  accurate 
results  than  the  electrometric  or  Potentiometric  Method.  The  principle 
of  this  method  has  already  been  explained  upon  page  154.  For  the 
degree  of  accuracy  usually  required  in  biochemical  or  physiological 
researches  the  apparatus  employed  by  Hoagland  (1)  and  illustrated  on 
page  272  (Fig.  10)  is  undoubtedly  the  simplest  and  most  convenient. 
For  solutions  not  containing  volatile  acids,  the  Cottrell  form  of  elec- 
trode as  modified  by  C.  L.  A.  Schmidt  is  the  best  (Fig.  11),  but  when 
the  fluid  to  be  investigated  contains  carbon  dioxide  which  would  be 
18 


274 


THE  PROPERTIES  OF  PROTOPLASM 


blown  out  by  a  continuous  stream  of  gas,  the  electrod  e  must  be  enclosed 
in  a  gas-tight  vessel  containing  hydrogen  and  the  fluid  to  be  investi- 
gated and  the  vessel  must  be  shaken  to  secure  continuous  contact  of 
the  electrode  with  hydrogen  so  as  to  maintain  its  saturation  (Fig.  12) . 
Certain  special  precautions  must  be  taken,  when  potentiometrically 
measuring  the  reaction  of  fluids  containing  proteins,  especially  that  of 
bringing  the  hydrogen  electrode  to  equilibrium  with  neutral  distilled 
water  before  immersing  it  in  the  protein  solution,  for  otherwise  the 
acid  reaction  of  the  platinum  due  to  the  great  excess  of  hydrogen  ions 
which  it  contains  will  precipitate  many  proteins  in  a  film  upon  its 
surface.  Foaming,  which  is  often  troubesome  in  protein  solutions, 
may  be  prevented  by  addition  of  a  few  drops  of  octyl  alcohol,  or  of  a 
mixture  of  amyl  alcohol  and  kerosine,  or  of  isoamyl  isovalerate. 


FIG.  12.— "Shaking"  hydrogen  electrode.     (After  Clark.) 

The  potentiometric  method  was  first  employed  for  the  estimation 
of  the  reaction  of  blood  by  Hoeber.  The  alkalinity  of  the  blood  which 
was  indicated  by  his  earliest  measurements  was  excessive,  owing  to  the 
fact  that  the  stream  of  hydrogen  employed  to  saturate  the  electrode 
blew  out  the  carbon  dioxide  which  in  circulating  blood  stands  in 
equilibrium  with  the  bicarbonates,  and  contributes  materially  to  the 
maintenance  of  neutrality.  Later  and  more  accurate  measurements 
by  Hoeber  and  many  others  are  unanimous  in  establishing  the  fact  that 
the  normal  reaction  of  the  blood  is  so  faintly  alkaline  as  to  approximate 
very  closely  to  neutrality.  Thus  at  absolute  neutrality,  as  in  neutral 
distilled  water,  the  hydrogen  and  hydroxyl  ions  are  equal  in  concentra- 
tion, namely  0.8  x  10~7  normal.  The  actual  hydroxyl  concentration 
in  the  blood  is  only  about  double  this,  namely  1.6  x  10~7  or  less  than 
one  five-millionth  normal  at  the  CO2-pressures  prevailing  in  the 
circulating  blood  (0.028  to  0.054  atmosphere). 


NEUTRALITY  OF  THE  TISSUES  AND  TISSUE-FLUIDS        275 

Another  method  which  has  been  extensively  employed  in  the  investi- 
gation of  the  reactions  of  blood-serum  and  of  other  tissue-fluids  is  the 
Indicator-method  of  Friedenthal  which  has  been  especially  applied  to 
these  investigations  by  Sorensen.  This  method  consists  in  adding 
to  the  fluid  under  investigation  a  number  of  different  indicators  known 
to  display  color-changes,  at  differing  hydrogen  or  hydroxyl  ion  concen- 
trations. The  same  indicators  are  also  added  to  a  series  of  mixtures 
of  monosodium  and  disodium  phosphate,  of  which  the  former  is  acid 
in  reaction  and  the  latter  alkaline.  The  hydrogen  ion  concentration  of 
all  possible  mixtures  of  these  salts  has  been  determined,  and  that 
mixture  which  yields  most  nearly  the  same  tints  with  indicators  as  the 
unknown  fluid  evidently  corresponds  to  it  in  hydrogen  ion  concentra- 
tion. This  method  is  not  applicable  to  a  highly  colored  fluid  such  as 
whole  blood  since  the  tints  of  indicators  are  not  accurately  appreciable 
in  such  a  fluid.  Furthermore  it  is  to  be  noted  that  the  indicators  most 
suitable  for  this  purpose  are  precisely  those  which  are  least  desirable 
for  the  ordinary  purposes  of  direct  titration,  because  the  best  indicator 
for  titration  is  that  which  displays  a  sharp  change  from  one  tint  to 
another  at  a  certain  reaction,  whereas  the  best  indicator  for  the  indirect 
method  of  titration  just  described  is  evidently  one  which  offers  a  large 
number  of  appreciable  changes  of  shade  or  tint  within  a  limited  range 
of  hydrogen  ion  concentrations.  The  most  suitable  indicator  for  the 
purposes  of  indirect  titration  within  the  range  of  reactions  commonly 
met  with  in  tissue-fluids,  is  Phenol  Sulphonphthalein.  Finally  it  should 
be  carefully  noted  that  the  choice  of  indicators  is  limited  to  those  which 
do  not  react  chemically  with  the  proteins  or  other  substances  commonly 
present  in  tissue-fluids.  A  variety  of  dyes  which  "are  commonly 
employed  as  indicators  in  direct  titration  are  unsuitable  for  our  purpose 
because  they  interact  with  proteins  and  the  compounds  which  are 
formed  do  not  change  color  at  the  hydrogen  ion  concentration  at  which 
the  free  dye  changes  color,  or  even  may  display  totally  different  colors 
from  those  which  the  free  dye  exhibits. 

By  these  various  methods  it  has  been  ascertained  that  not  only  is  the 
blood  of  all  vertebrates  very  nearly  neutral  in  reaction,  but  almost  all 
of  the  tissue-fluids  are  also  approximately  neutral.  Thus  the  Pan- 
creatic Juice,  the  most  alkaline  of  body-fluids,  contains  5  x  10~9  H+, 
corresponding  to  an  alkalinity  of  13  x  10~7  OH~  or  a  little  over  one 
millionth  normal.  Hitherto,  according  to  Friedenthal,  no  animal 
fluid  has  been  found  which  contains  less  than  10~10H+,  that  is,  more 
than  about  10Q6000  normal  OH~. 

Now  the  neutrality  of  the  blood  is  maintained  with  extraordinary 
exactitude  despite  the  fact  that  a  large  proportion  of  the  products  of 
metabolism  are  acid  in  reaction  and  are  washed  out  of  the  tissues  in 
which  they  are  formed,  into  the  blood.  The  products  of  muscular 
activity  include  carbon  dioxide,  lactic  acid  and  acid  phosphates,  and 
the  muscular  exertion  which  is  involved,  for  example,  in  climbing  a 
steep  hill  involves  the  expenditure  of  a  very  considerable  number  of 


276  THE  PROPERTIES  OF  PROTOPLASM 

foot-pounds  of  energy,  and  the  oxidation  of  a  correspondingly  large 
quantity  of  carbohydrate  material,  of  which  the  carbon  is  converted 
ultimately  into  carbon  dioxide,  which  is  carried  to  the  lungs  through 
the  mediation  of  the  blood.  Yet  while  this  large  production  of  acid 
products  may  cause  some  slight  distress  of  breathing  in  the  unaccus- 
tomed individual,  it  barely  perceptibly  modifies  the  reaction  of  the 
blood.  The  intravenous  injection  of  large  quantities  of  acid  produces 
an  altogether  disproportionately  small  effect  upon  the  alkalinity  of  the 
blood.  In  Diabetes  the  faulty  oxidation  of  fats  produces  a  quantity  of 
non-volatile  acids  which  cannot  be  discharged  as  carbon  dioxide  is 
discharged,  through  the  respiratory  epithelium  of  the  lungs,  and  yet 
in  many  cases  of  advanced  diabetes  the  reaction  of  the  blood  is  only 
very  slightly  affected  so  that  even  in  diabetic  coma  the  acidity  of  the 
blood  is  only  raised  to  1  x  10~7  normal  H+,  a  reaction  which  would  be 
communicated  to  a  hundred  liters  of  neutral  distilled  water  by  the 
addition  of  a  single  drop  of  normal  acid  solution. 

The  mechanism  whereby  this  extraordinary  stability  of  reaction  is 
attained  is  a  dynamic  equilibrium  which  involves  a  variety  of  coordi- 
nated factors.  Thus  the  kidneys  assist  in  removing  excess  of  acids  by 
excreting  a  predominance  of  acid  salts  and  of  non-volatile  acids.  The 
lungs  are,  however,  the  most  important  organs  of  acid-elimination, 
since  they  contribute  to  the  reduction  of  the  hydrogen  ion  concentra- 
tion of  the  blood  by  permitting  the  escape  of  carbon  dioxide.  On  the 
other  hand  the  tissues  themselves  can  contribute  to  the  neutralization 
of  injurious  excess  of  hydrogen  ions  by  arresting  the  formation  of  urea 
from  protein  nitrogen  at  the  intermediate  stage  of  ammonia,  the 
ammonium  salts  of  the  excessive  acids  being  excreted  as  such  in  the 
urine.  Hence,  in  Acidosis  such  as  that  encountered  in  diabetes  and 
in  many  toxemias,  an  unusual  quantity  of  Ammonia  appears  in  the 
urine. 

The  prime  agent  in  accomplishing  the  regulation  of  the  reaction  of 
the  tissues  and  tissue-fluids  is,  however,  the  blood  itself.  This  may  very 
readily  be  perceived  by  comparing  the  relative  powers  of  blood  and  of 
distilled  water  or  sodium  chloride  solution  to  neutralize  acids.  If 
two  indicators  be  chosen  which  change  color  at  differing  hydrogen  ion 
concentrations,  and  distilled  water  and  blood-serum  respectively  be 
neutralized  first  to  one,  and  then  to  the  other  indicator,  the  difference 
between  the  two  /iters  will  be  extremely  small  in  the  case  of  distilled 
water  and  of  very  considerable  magnitude  in  the  case  of  the  blood- 
serum.  It  can  be  shown  in  fact,  that  provided  the  carbon  dioxide 
tension  be  maintained  at  the  levels  which  prevail  in  circulating  blood, 
one  hundred  volumes  of  blood  of  normal  reaction  can  neutralize  no  less 
than  125  volumes  of  ^  hydrochloric  acid  before  attaining  the  hydro- 
gen ion  concentration  of  advanced  acidosis,  namely  l.OOx  10~7  at  38°. 
This  would  be  equivalent,  in  a  man  whose  circulation  contains  five 
liters  of  blood,  to  the  neutralization  of  over  six  liters  of  or  six  hun- 


NEUTRALITY  OF  THE  TISSUES  AND  TISSUE-FLUIDS        277 

dred  cubic  centimeters  of  ^  acid.  In  the  body,  it  must  be  remembered, 
this  remarkable  neutralizing-power  of  the  blood  is  assisted  by  the  added 
ventilation  of  carbon  dioxide  from  the  lungs,  which  occurs  in  conse- 
quence of  the  Dyspnea  or  rapid  breathing  which  results  from  a  slight 
decrease  of  the  alkalinity  of  the  blood,  and  by  the  excretion  of  acid 
salts  by  the  kidneys  and  by  the  production  of  ammonia  from  the  tissues. 
The  origin  of  the  neutralizing-power  of  the  blood  is  threefold :  in  the 
first  place  the  Bicarbonates  of  the  blood  are  capable  of  neutralizing 
large  quantities  of  acid  without  any  great  change  in  the  hydrogen  ion 
concentration  by  undergoing  the  reactions : 

NaHCOs     +     HA      =     NaA     +     H2CO3 
H2CO3      =     H2O      +     CO2 

Thus  if  the  acid  HA  is  strongly  dissociated,  the  effect  of  these  trans- 
formations is  to  replace  it  by  the  exceedingly  weakly  dissociated  car- 
bonic acid  or  by  the  neutral  gas  carbon  dioxide.  In  a  similar  manner 
the  Phosphates  of  the  blood  contribute  to  maintain  neutrality  by  under- 
going the  reaction : 

Na2HPO4     +     HA      =     NaH2PO4     +     NaA 

whereby  the  strongly  dissociated  acid  HA  is  replaced  by  the  faintly 
acid  salt,  monosodium  phosphate.  Finally  the  Protein  Salts  in  the 
blood  also  assist  in  the  preservation  of  neutrality  by  entering  into 
reactions  of  the  type: 

Na  Protein     +     HA      =     H  Protein      +     NaA 

the  strong  acid  being  in  this  instance  replaced  by  practically  neutral 
uncombined  protein. 

Of  these  three  agencies  the  bicarbonates  are  quantitatively  much 
the  most  important.  This  arises  from  their  abundance  in  plasma  and 
also  from  the  fact  that  the  dissociation-constant  of  carbonic  acid,  or 
proportion  of  hydrogen  ions  to  undissociated  acid  in  the  reaction  of 
dissociation : 

H2CO3     ^±    H+     +     HCO-3 

is  very  nearly  equal  to  the  hydrogen  ion  concentration  in  distilled  water 
at  absolute  neutrality  (0.8  x  10~7  normal).  Now  L.  J.  Henderson  has 
shown  that  the  rate  of  change  in  the  alkalinity  or  acidity  of  a  solution 
of  an  acid  when  alkalies  or  acids  are  added  to  it  is  a  minimum  when  the 
dissociation-constant  of  the  acid  is  of  this  magnitude.  He  illustrates 
this  principle  by  the  following  table,  showing  the  amount  of  tenth 
normal  alkali  required  to  secure  a  definite  but  arbitrarily  chosen  change 
in  alkalinity  when  added  to  equal  amounts  of  the  undermentioned  acids : 

Dissociation-constant.  Cubic  centimeters  of 

Acid.                                                                   X10~7  alkali  required. 

Phenol         0.0013  0.01 

Boric  acid 0.017  0.08 

Hydrogen  sulphide 0.57  1.10 

Monosodium  phosphate 2.0  1 . 00 

Carbonic  acid 3.0  0.72 

Picolinic  acid 18.0  0.10 

Acetic  acid                                                  .180.0  0.03 


278 


THE  PROPERTIES  OF  PROTOPLASM 


The  ability  of  sodium  bicarbonate,  in  equilibrium  with  carbonic 
acid,  to  maintain  the  neutrality  of  its  solutions  is  strikingly  illustrated 
by  Henderson  in  the  following  way :  "  Suppose,  for  example,  a  solution 
of  100  liters  containing  one  kilogram  of  sodium'  bicarbonate  in 
equilibrium  with  an  atmosphere  containing  one  gram  of  carbon 
dioxide  per  liter.  Let  hydrochloric  acid  be  added  in  successive  small 
portions  to  the  solution.  Further,  let  the  solution  be  constantly  stirred 
and  shaken,  and  let  the  experiment  be  conducted  slowly,  so  that  there 
shall  always  be  equilibrium  between  the  carbonic  acid  in  the  solution 
and  in  the  atmosphere.  Further,  let  the  temperature  be  such  that  the 
absorption-coefficient  of  carbon  dioxide  shall  be  1.000.  Then  the 
successive  states  of  the  solution  will  be  approximately  as  recorded  in 
the  following  table. 


HCl  added, 
grams. 

Ratio  of  H2CO3 
to  NaHCOs. 

H+  normal. 

OH    normal. 

Relative 
acidity. 

Relative 
alkalinity. 

0    . 

2.  27  to  11.90 

0.000000057 

0.000000176 

0.57 

1.76 

10    . 

2.  27  to  11.50 

0.000000059 

0.000000170 

0.59 

1.70 

50    . 

2.  27  to  10.00 

0.000000068 

0.000000147 

0.68 

1.47 

100    . 

2.  27  to    8.20 

0.000000083 

0.000000120 

0.83 

1.20 

150    . 

2.  27  to    6.30 

0.000000108 

0.000000093 

1.08 

0.93      ' 

200    . 

2.  27  to    4.40 

0.000000154 

0.000000065 

1.54 

0.65 

250    . 

2.  27  to    2.60 

0.00000026 

0.000000039 

2.60 

0.39 

300    . 

2.  27  to    0.68 

0.0000010 

0.000000010 

10. 

0.10 

310    . 

2.27to    0.31 

0.0000022 

0.0000000045 

22. 

0.045 

318     . 

ex 

0.00026 

0.00000000039 

260. 

0.0039 

320    .      . 

0.00045 

0.00000000022 

450. 

0.0022 

330    .      . 

0.  0027 

0.000000000037 

2700. 

0.00037 

"From  the  beginning  of  the  experiment  until  almost  250  grams 
of  hydrochloric  acid  have  been  added,  neither  alkalinity  nor  acidity 
is  double  in  intensity  the  values  which  obtain  in  a  perfectly  neutral 
solution."  "Such  close  approach  to  neutrality  can  be  attained  with 
pure  water  only  after  elaborate  and  very  difficult  purification,  yet  in 
the  presence  of  carbonic  acid  it  is  the  natural  condition." 

In  laboratory-glassware  a  mixture  of  disodium  and  monosodium 
Phosphates  would  perhaps  be  almost  as  efficient  as  sodium  bicarbonate 
in  preserving  neutrality.  In  the  body,  however,  they  are  not  so 
efficient  as  the  bicarbonates  because  in  the  first  place  they  are  not 
nearly  so  abundant  and  in  the  second  place  the  elimination  of  the  acid 
phosphates  which  are  formed  in  the  neutralization  of  acids  has  to  take 
place  by  the  relatively  slow  and  roundabout  channel  of  the  kidneys, 
while  the  elimination  of  carbon  dioxide  takes  place  rapidly  through 
ventilation  from  the  lungs. 

Direct  determinations  by  the  potentiometric  method  have  shown 
that  the  proteins  contribute  just  about  one-fifth  of  the  neutraliz ing- 
power  of  the  blood.  In  the  tissues  their  proportional  importance 
in  maintaining  neutrality  is  probably  greater,  because  they  are  present 
therein  in  higher  concentration  than  they  are  in  the  blood. 


NEUTRALITY  OF  THE  TISSUES  AND  TISSUE-FLUIDS        279 

Solutions  such  as  those  of  sodium  bicarbonate,  disodium  phosphate 
or  sodium  proteinate  which  conserve  the  neutrality  of  the  water  in 
which  they  are  dissolved  are  very  frequently  designated  "Buffer- 
solutions"  from  the  resemblance  of  their  action  obliterating  rapid 
changes  of  hydrogen  concentration  to  the  action  of  a  buffer  on  a  vehicle 
in  obliterating  dangerously  sudden  changes  of  velocity  of  motion. 
Buffer-solutions  are  frequently  employed  now,  and  must  necessarily 
be  more  and  more  widely  employed,  wherever  stable  conditions  of 
environment  are  requisite,  as  in  bacterial  cultures,  cultures  of  living 
tissue  in  vitro,  aquarium-media  for  marine  or  fresh-water  organisms, 
and  artificial  circulating  media. 

An  estimation  of  the  very  greatest  importance  in  all  disease-condi- 
tions or  metabolic  disturbances  which  involve  Acidosis  is  that  of  the 
Alkali-reserve  or  neutraliz ing-power  of  the  blood.  When  large  quanti- 
ties of  "fixed"  i.e.,  non- volatile  acids  are  thrown  into  the  blood  the 
sodium  with  which  they  combine  is  rendered  unavailable  for  neutraliz- 
ing other  portions  of  acid  or  for  binding  carbon  dioxide.  The  alkali- 
reserve  in  such  cases  is  diminished  and  the  ability  of  the  blood  to 
maintain  its  neutrality  is  proportionately  impaired.  A  low  alkali- 
reserve  is  therefore,  in  general,  a  relatively  hazardous  condition. 

Various  methods  have  been  proposed  for  measuring  the  alkali-reserve, 
the  majority  depending  upon  the  fact  that  as  the  sodium  bicarbonate 
of  the  blood  has  been  diminished  and  the  uncombined  carbon  dioxide 
stands  in  almost  constant  proportion  to  it,  the  carbon  dioxide  obtain- 
able from  the  blood  by  a  standard  procedure  is  diminished.  The 
method  suggested  by  Van  Slyke,  and  now  employed  very  widely, 
consists  in  taking  a  sample  of  blood  from  a  vein  in  the  forearm  and 
introducing  it  into  a  vessel  filled  with  the  alveolar  air  of  the  patient 
obtained  by  breathing  and  rebreathing  into  the  vessel  several  times. 
The  blood  is  then  shaken  up  with  the  alveolar  air  to  bring  it  into  equi- 
librium with  the  carbon  dioxide  contained  therein  and  a  measured 
sample,  without  loss  of  carbon  dioxide,  is  introduced  into  a  special 
form  of  gas-burette  (Fig.  13)  and  acidified  with  sulphuric  acid  to 
decompose  the  bicarbonates.  The  chamber  containing  the  sample 
is  now  evacuated  by  means  of  a  column  of  mercury  and  the  gas  which 
is  evolved  is  measured  at  atmospheric  temperature  and  pressure. 

An  alternative  and  perhaps  preferable  method  which  is,  however, 
somewhat  less  simple  to  manipulate,  consists  in  directly  analyzing  the 
carbon  dioxide  content  of  Alveolar  Air  obtained  by  rebreathing  into 
a  closed  vessel.  When  the  alkali-reserve  is  low,  the  carbon-dioxide 
content  of  the  blood  being  diminished,  the  carbon-dioxide  output 
through  the  lungs  and  the  partial  pressure  of  carbon  dioxide  in  the 
alveolar  air  are  correspondingly  diminished. 

Another  feasible  method  of  measuring  the  alkali-reserve,  or,  which 
comes  to  the  same  thing,  the  Neutralizing-power  of  tissue-fluids  is  that 
which  has  been  employed  by  Marshall  in  the  analysis  of  Saliva.  The 


280 


THE  PROPERTIES  OF  PROTOPLASM 


Petition  1 


\rnrn.  bore 


Position  3 
Is  6ocm  below 
position  £ 


FIG.   13. — Van  Slyke's  apparatus  for  the  determination  of  carbon  dioxide  in  blood. 

(After  Van  Slyke.) 


NEUTRALITY  OF  THE  TISSUES  AND  TISSUE-FLUIDS        281 

various  samples  of  saliva  having  been  brought  to  a  common  reaction 
on  the  alkaline  side  of  absolute  neutrality  (neutrality  to  phenolphtha- 
lein),  the  quantity  of  acid  is  estimated,  by  direct  titration,  which  is 
necessary  to  bring  the  reaction  of  the  fluid  to  an  arbitrarily  chosen 
reaction  on  the  acid  side  of  neutrality  (neutrality  to  paranitrophenol) . 
The  measurement  is  in  fact  analogous  to  that  employed  by  Henderson 
in  estimating  the  power  of  different  acids  to  maintain  the  neutrality 
of  their  solutions.  Of  course,  to  obtain  physiologically  interpretable 
results  with  blood-serum  it  would  be  necessary  to  carry  out  the  titra- 
tions  under  a  standard  partial  pressure  of  carbon  dioxide,  for  example 
in  a  vessel  filled  with  alveolar  air.  The  choice  of  indicators  is  limited 
when  the  fluid  under  investigation  is  even  faintly  tinged  with  color. 
For  example  the  faint  yellowish  tinge  of  blood-serum  interferes  with 
the  sharpness  of  the  end-point  with  paranitrophenol. 

We  thus  see  that  by  a  variety  of  interlocking  mechanisms,  consisting 
in  every  instance  of  dynamic  equilibria,  the  tissue-fluids  of  the  higher 
animals,  which  are  to  their  individual  cells  the  external  media  in  which 
they  live,  are  kept  extraordinarily  constant  in  concentration,  composi- 
tion of  mineral  constituents,  and  hydrogen  ion  concentration. 

The  very  great  susceptibility  of  most  of  the  chemical  reactions 
which  are  involved  in  life-phenomena  to  slight  changes  of  reaction, 
may  very  readily  be  seen  to  involve  relative  stability  of  reaction  as  a 
requisite  to  the  orderly  performance  of  life-processes.  It  is  in  fact  an 
almost  universal  rule,  in  the  words  of  Loeb,  that  "  life-phenomena  occur 
in  a  neutral  liquid. "  The  ocean  which  is  the  original  home  of  life,  is, 
thanks  to  the  presence  of  bicarbonates  and  phosphates,  a  "buffer"- 
solution  and  nearly  neutral  in  reaction  despite  the  life  which  swarms 
therein.  According  to  Palitzsch  the  extreme  variation  in  the  hydrogen 
ion  concentration  of  the  ocean  is  from  1.1  x  10~8  N  to  0.45  x  10~8  N  H+, 
corresponding  to  an  exceedingly  faint  alkalinity  of  the  order  of  that 
found  in  the  blood  of  mammals.  In  a  very  few  instances  only  does 
life  subsist  in  a  medium  which  deviates  far  from  neutrality.  When 
secreting  gastric  juice,  in  the  absence  of  neutralizing  substances,  the 
cells  of  the  gastric  mucosa  are  bathed  on  the  side  toward  the  lumen 
of  the  stomach  by  a  fluid  which  may  attain  an  acidity  or  hydrogen  ion 
concentration  due  to  hydrochloric  acid  of  no  less  than  one  hundredth 
normal,  or  ten  thousand  times  the  acidity  which  would  correspond  to 
the  alkalinity  of  pancreatic  juice.  The  "salivary  glands"  of  certain 
carnivorous  molluscs,  which  probably  correspond  in  function  to  the 
gastric  glands  in  mammalia,  similarly  secrete  an  acid  juice  in  which 
the  high  hydrogen  ion  concentration  is  attributable  to  Sulphuric  Acid. 

With  such  rare  exceptions,  exhibited  only  by  highly  specialized  and 
adapted  cells,  the  immediate  environment  in  which  living  matter 
subsists  is  extremely  invariable  in  certain  physical  characteristics, 
and  this  invariability  which  is  essential  to  the  normal  occurrence  of 
life-phenomena,  is  brought  about  through  the  interplay  of  unique 


282  THE  PROPERTIES  OF  PROTOPLASM 

physical  and  chemical  properties  which  are  possessed  by  water  and 
carbon  dioxide.  Even  the  additional  stability  of  the  environment  of 
life  which  is  brought  about  by  the  maintenance  of  constant  temperature 
in  the  homoiothermal  animals,  is  dependent  upon  the  unique  specific 
heat  of  water.  The  fitness  of  our  environment  for  life  is  therefore 
essentially  dependent  upon  these  substances.  As  Henderson  has 
pointed  out,  it  is  not  that  living  matter  has  become  adapted  in  an 
evolutionary  sense  to  this  medium,  although  specific  organs  concerned 
in  the  maintenance  of  the  stability  of  the  environment  in  higher 
organisms,  such  as  the  kidney,  may  have  been  subject  to  evolutionary 
adaptation.  For  the  environment  or  the  conditions  from  which  it 
inevitably  arose  long  antedated  life  itself,  and  the  earliest  forms  of 
life  must  have  been  fitted  to  this  environment  no  less  exactly  than  the 
later.  A  direct  chemical  interrelationship  between  life  phenomena 
and  the  particular  type  of  environment  in  which  they  occur  is  thus 
indicated. 

It  is  somewhat  idle  to  speculate  whether  or  not  life  could  subsist 
in  some  quite  different  environment  with  some  other  element  such  as 
silicon  or  boron  as  a  base  instead  of  carbon.  Such  "life,"  if  it  could 
correctly  be  so  called,  lies  of  necessity  outside  our  experience.  But 
the  absolute  dependence  of  life  as  we  know  it  upon  water  in  the  liquid 
form  and  carbon  dioxide  in  the  gaseous  form,  renders  the  temperature 
limits  between  which  life  can  subsist  excessively  narrow  in  comparison 
with  the  vast  range  of  temperatures  found  in  the  various  portions  of 
our  universe.  Of  the  6500  degrees  which  separate  the  temperature  of 
interstellar  space  from  that  of  the  surface  of  the  sun,  only  65°  or  one 
per  cent,  of  the  total  range  is  suitable  for  the  occurrence  of  life-pheno- 
mena. In  view  of  this  exceedingly  narrow  margin  upon  which  life 
precariously  depends,  the  probability  of  its  presence  in  any  other  of  the 
bodies  in  our  solar  system  must  be  regarded  as  exceedingly  small. 
Concerning  the  possibility  of  life  in  other  suns  or  planets  which  may 
be  associated  with  them,  we  are  of  course  in  complete  ignorance,  but 
Arrhenius  has  put  forward  the  interesting  hypothesis  that  life  may  be 
transmissable,  in  the  latent  form  which  is  embodied  in  bacterial  spores, 
from  one  part  of  the  universe  to  another  in  association  with  cosmic 
dust.  Bacterial  Spores  have  been  experimentally  shown  to  be  exceed- 
ingly resistant  to  desiccation  and  low  temperatures,  retaining  their 
ability  to  give  rise  to  functionally  active  protoplasm  so  soon  as  they 
encounter  a  favorable  environment.  The  computations  of  Arrhenius 
show  that  the  known  properties  of  certain  bacterial  spores  are  not 
inconsistent  with  the  view  that  they  might  survive  a  journey  through 
space,  impelled  by  light-pressure,  from  one  solar  system  to  another. 
If  this  view  be  correct,  then  the  existence  of  life  in  any  part  of  the 
universe  might  sow  the  whole  with  seeds  ready  to  develop  at  any  mo- 
moment  at  which  the  environment  of  a  particular  cosmic  body  becomes 
suitable  for  the  maintenance  of  the  processes  of  functionally  active  life. 


NEUTRALITY  OF  THE  TISSUES  AND  TISSUE-FLUIDS         283 


REFERENCES. 
GENERAL: 

Hamburger:     Osmotische  Druck  und  lonenlehre.     Wiesbaden,  1907. 

Hoeber:  Physikalische  Chemie  der  Zelle  und  der  Gewebe:  Leipzig.  Fourth 
edition. 

Loeb,   J.:     Studies  in  General   Physiology.     Chicago,    1905. 

Loeb,  J.:     The  Dynamics  of  Living  Matter.     New  York,  1906. 

Philip:     Physical  Chemistry,  its  Bearing  on  Biology  and  Medicine.    London,-  1914. 

Michaelis:     Die  Wasserstoffionen  Konzentration.     Berlin,   1914. 
OSMOTIC  PRESSURE  OF  TISSUE-FLUIDS: 

Fano  and  Bottazzi:     Arch.  Ital.  di  Biol.,  1896,  26,  p.  45. 

Bottazzi  and  Ducceschi:     Ibid.,  1896,  26,  p.  161. 

Bottazzi:     Ibid.,  1897,  28,  p.  61.     Arch,  di  Fisiol.,  1906,  3,  pp.  416  and  547. 

Bottazzi  and  Gabrieli:     Arch.  Int.  de  Physiol.,  1905-6,  3,  p.  156. 

Bottazzi:     Ergeb.  der  Physiol.,  1908,  7,  p.  161. 

Atkins:     Biochem.  Jour.,  1909,  4,  p.  480. 

Jona:     Ibid.,  1912,  6,  p.  130. 
OSMOTIC  PRESSURE  OF  CELL-CONTENTS: 

Hedin:  Skand.  Arch.  f.  Physiol.,  1895,  5,  pp.  207,  238,  377.  Pfliiger's  Arch.,  1897, 
68,  p.  229;  1898,  70,  p.  525. 

Koeppe:     Arch.  f.  Anat.  u.  Physiol.,  Abt.,  1895,  p.  154. 
COMPOSITION  OF  THE  MINERAL  CONSTITUENTS  OF  TISSUE-FLUIDS: 

Macallum:     The  Ancient  Factors  in  the  Relations  between  the  Blood-plasma  and 

the  Kidneys,  Trans,  of  the  Coll.  of  Phys.  of  Philadelphia,  1917 
NEUTRALITY  OF  THE  TISSUES  AND  TISSUE-FLUIDS: 

Hoeber:     Pfliiger's  Arch.,  1900,  81,  p.  522;  1903,  99,  p.  572. 

Friedcnthal:     Zeit.  f.  allg.  Physiol.,  1902,  1,  p.  56;  1904,  4,  p.  44. 

Farkas:  Arch.  f.  Anat.  und  Physiol.,  Abt.  Supp.,  1903,  p.  517;  Pfliiger's  Arch., 
1903,  98,  p.  551. 

Fraenkel:     Ibid.,  1903,  96,  p.  601. 

v.  Szily:     Ibid.,  1906,  115,  pp.  72  and  82. 

Robertson:     Jour.  Biol.  Chem.,  1909-10,  7,  p.  351. 

Hasselbalch:     Biochem.  Zeit.,  1910,  30,  p.  317;  1913,  49,  p.  451. 

Hasselbalch  and  Tundsgaard:     Ibid.,  1912,  38,  p.  77. 

Henderson:     The  Fitness  of  the  Environment,  New  York,  1913. 
METHOD  OF  ESTIMATING  THE  REACTION  OF  TISSUE-FLUIDS: 

Schmidt:     University  of  California,  Physiology  Pub.,  1909,  3,  p.  101. 

Sorensen:     Ergeb.  d.  Physiol.,  1912,  12,  p.  393. 

Sharp  and  Hoagland:     Jour.  Agr.  Res.,  1916,  7,  p.  123. 

Clark  andLubs:     Jour.  Bact.,  1917,  2,  pp.  I,  109,  191. 

Schmidt  and  Hoagland:  Table  of  PH,  H+  and  OH~  values  Corresponding  to  Electro- 
motive Forces  Determined  in  Hydrogen  Electrode  Measurements,  University 
of  California  Publications,  Physiology,  1919,  5,  p.  23  (consult  for  literatxire  on 
gas-chain). 

Baker  and  Van  Slyke:     1918,  35,  p.  137. 
METHODS  OF  ESTIMATING  THE  ALKALI-RESERVE: 

Henderson:  The  Excretion  of  Acid  in  Health  and  Disease,  Harvey  Lectures,  1  Oth 
Ser.,  Philadelphia,  1914-15,  p.  132. 

Levy  and  Rowntree:     Arch.  Int.  Med.,  1916,  17,  p.  525. 

Sellards:  The  Principles  of  Acidosis  and  Clinical  Methods  for  its  Study.  Cam- 
bridge, Mass.,  1917. 

Van  Slyke:     Jour.  Biol.  Chem.,  1917,  30,  pp.  289,  347. 


CHAPTER  XIII. 

PROPERTIES  CONFERRED  BY  THE  COLLOIDAL  CON- 
STITUENTS: STRUCTURE  AND  CONSISTENCY. 

THE  EMULSION-STRUCTURE  OF  PROTOPLASM. 

One  of  the  most  important  aspects  of  the  relationship  of  the  Lipoids 
to  the  properties  and  behavior  of  protoplasm  is  that  arising  out  of  the 
marked  effect  upon  the  tension  of  protoplasmic  surfaces  which  the 
lipoids  and  their  decomposition-products  are  capable  of  bringing  about. 
The  Surface-tension  of  the  interface  between  water  and  gas,  or  an 
immiscible  fluid  or  solid,  is  very  markedly  reduced  by  Oils,  Fatty 
Acids  or  Soaps,  and  this  fact  contributes  in  the  first  place  to  the  deter- 
mination of  the  distribution  of  these  substances  in  the  cell,  and  in  the 
second  place  to  the  stability  of  the  emulsified  substances  in  living  cells, 
which,  despite  their  immiscibility  in  water,  remain  suspended  in  the 
form  of  stable  emulsions  within  the  material  composing  the  protoplasm. 

The  distribution  of  soluble  fatty  materials  in  the  cell,  such  as  the 
Lecithins  must  be  considerably  influenced  by  the  extent  and  variety 
of  the  surfaces  which  are  presented  by  the  sponge-  or  foam-like  structure 
of  protoplasm.  The  reason  for  this  is  that  all  those  substances  which 
reduce  superficial  tensions  also  tend,  if  possible,  to  become  concen- 
trated upon  any  surfaces  presented  to  them.  This  is  very  strikingly 
shown,  for  example,  in  a  classical  experiment  adduced  by  J.  J.  Thomson. 
If  a  deeply  colored  solution  of  Potassium  Permanganate  be  passed 
through  a  long  column  of  well  washed  and  finely  ground  quartz-sand, 
the  first  few  drops  of  fluid  which  percolate  through  the  column  will 
be  found  to  be  colorless,  the  whole  of  the  permanganate  in  this  first 
quantum  having  been  abstracted  from  the  solution  by  the  surfaces 
over  which  it  has  passed,  not  because  of  any  chemical  interaction 
between  the  sand  and  the  reagent,  but  in  consequence  of  the  reduction 
of  the  tension  of  the  water-sand  interface  by  the  permanganate. 
Similarly,  if  aqueous  solutions  of  Saponins  or  of  Bile-salts  be  shaken 
up  with  petroleum-oils,  the  dissolved  substance  will  be  found  to  have 
become  concentrated  at  the  surface  of  the  oil-drops,  and  in  the  foam 
which  forms  when  saponin  solutions  are  shaken  in  air,  the  saponin  is 
more  concentrated  than  it  is  in  the  body  of  the  liquid. 

The  mechanism  of  this  retention  of  dissolved  substances  by  sur- 
faces is  as  follows:  In  the  accompanying  diagram  (Fig.  14)  of  a 
spherical  droplet  partially  enclosed  by  a  layer  of  molecules  which  coat 
it  and  separate  it  excepting  at  the  gap  A  from  direct  contact  with 
the  surrounding  medium,  if  the  enveloping  molecules  reduce  the 


EMULSION-STRUCTURE  OF  PROTOPLASM  285 

tension  of  the  interface  between  the  drop  and  the  medium  in  which  it 
is  suspended,  it  is  evident  that  the  tension  of  the  exposed  gap  in  the 
surface  will  be  greater  than  the  tension  of  the  covered  portions  of  the 
surface.  The  two  portions  of  the  surface  will  be  pulling  unequally, 
therefore,  and  unbalanced  excess  of  tension  will  exist  at  the  gap  in  the 
sense  indicated  by  the  arrows,  and  the  tendency  of  this  tension  in  the 
case  of  a  gap  of  molecular  dimensions  will  obviously  be  to  draw  together 
the  edges  of  the  enveloping  film  and  reduce  the  tension  of  all  parts  of 
the  surface  to  a  uniform  value.  Any  molecules  of  such  a  substance 
coming  into  contact  with  the  surface  will  therefore  tend  to  be  held  or 
"trapped"  there,  and  since,  in  the  course  of  the  fortuitous  motions  of 
the  dissolved  molecules,  a  very  large  number  must  repeatedly  come  into 
contact  with  any  surfaces  exposed  to  a  solution,  the  accumulation  of 
adhering  molecules  will  continue  until  the  droplet  becomes  covered  by 
a  layer  of  such  thickness  that  the  molecular  attraction  between  the 
underlying  molecules  of  the  drop  and  those  of  the  surrounding  medium 
becomes  inappreciable  owing  to  the  distance  through  which  it  has  to 
be  exerted.  This  will  occur  when  the  thickness  of  the  film  is  of  the 
order  of  that  of  a  soap-bubble  just  before  it  bursts,  namely  about  one 

two  millionth  of  a  millimeter. 

A 


FIG.  14. — Illustrating  the  tendency  of  a  lipoidal  layer  at  the  interface  of  two  aqueous 
phases  to  repair  itself  when  broken. 

The  extent  to  which  the  Surface-tension  of  water  is  reduced  by 
lipoidal  substances  and  soaps  may  be  inferred  from  the  following 
results  reported  by  Lord  Rayleigh.  The  measurements  refer  to  an 
air-water  interface. 

Dynes  per 
linear  c.m. 

Tension  of  pure  water1 74 

Tension  of  greasy  water 33 

Tension  of  water  saturated  with  olive  oil 41 

Tension  of  water  saturated  with  sodium  oleate 25 

The  trace  of  Olive  Oil  which  dissolves  in  water,  therefore,  reduces 

the  tension  of  the  air-water  interface  to  one-half  its  normal  magnitude. 

The  lipoids  in  the  cell  are  present  partly  in  soluble  forms,  such  as 

^The  tension  of  the  air- water  interface  is  usually  considered  to  be  81  dynes  per 
square  centimeter  which  is  the  estimate  of  Quincke.  The  estimate  of  Lord  Rayleigh 
who  employed  very  refined  method  of  measurement  is  more  probably  correct.  In  any 
case,  all  of  the  estimates  having  been  made  by  the  same  method,  they  are  comparable 
with  one  another. 


286     PROPERTIES  CONFERRED  BY  COLLOIDAL  CONSTITUENTS 

the  Lecithins,  partly  in  a  very  finely  emulsified  form,  the  individual 
particles  being  of  ultramicroscopic  dimensions,  and  partly  in  a 
coarsely  emulsified  form  such  as  that  found  in  fatty  connective  tissues. 
The  presence  of  a  large  proportion  of  ultramicroscopically  divided  fat 
is  shown  by  the  high  fat-content  of  many  tissues  in  which  microscopical 
examination  after  appropriate  staining  fails  to  reveal  the  presence  of 
visible  fat-globules.  Under  certain  conditions,  especially  in  Phosphorus- 
poisoning  and  in  Anaphylactic  Shock,  the  ultramicroscopic  particles  in 
certain  tissues  coalesce  to  form  coarse  emulsions  and  the  particular 
tissues  affected,  as  for  example  the  liver  in  phosphorus-poisoning,  are 
then  easily  seen  to  be  heavily  infiltrated  with  fat.  Direct  analysis, 
has  shown  that  in  such  cases  the  fat-content  of  the  tissues  is  neverthe- 
less normal,  in  other  words  the  normal  liver-cell  contains  just  as  much 
fat  as  the  liver-cell  which  has  undergone  fatty  degeneration  in  conse- 
quence of  phosphorus-poisoning,  but  in  the  normal  cell  the  Emulsifica- 
tion  of  the  fat  is  so  thorough  that  the  greater  part  of  the  fat  is  present 
in  particles  too  small  to  be  visible  under  the  microscope.  The  soluble 
lipoids  and  the  soaps  and  other  substances  which  reduce  the  tension  of 
an  oil-water  interface  are  probably  in  large  proportion  concentrated 
at  the  extensive  surfaces  which  arise  from  this  subdivision. 

The  Emulsification  of  fats  in  water  is  greatly  facilitated  by  the 
presence  in  the  water  of  a  substance  which  reduces  the  interfacial 
tension,  provided  that  at  the  same  time  the  substance  forms  a  viscous 
or  sparingly  soluble  coating  over  the  oil-droplets  which  retards  their 
coalescence  when  they  come  fortuitously  into  contact  with  one  another. 
We  have  already  had  occasion  to  dwell  upon  the  importance  of  soaps 
and  of  the  bile-salts  in  bringing  about  the  emulsification  of  the  fats  in 
the  diet  prior  to  their  hydrolysis  by  the  digestive  enzymes. 

When  olive  oil  is  shaken  up  with  pure  water  little  or  no  emulsification 
occurs.  Even  when  the  mixture  has  been  very  thoroughly  shaken, 
the  oil  and  water  separate  completely  within  a  comparatively  brief 
period.  If,  however,  a  little  sodium  carbonate  or  hydroxide  be  added 
to  the  water  in  order  to  form  soap  with  the  trace  of  fatty  acid  which  oil 
contains,  the  effect  of  shaking  the  mixture  is  now  very  different.  A 
milky  or  creamy  emulsion  is  formed  with  comparatively  little  expendi- 
ture of  mechanical  effort  in  shaking,  and  no  separation  of  the  two 
fluids  will  occur  even  after  long  intervals  of  time.  The  emulsifying 
action  of  alkalies  is  also  strikingly  illustrated  by  floating  drops  of  Olive 
Oil  upon  distilled  water  and  one  per  cent,  sodium  carbonate  solution 
respectively.  In  the  latter  case  the  oil-droplet  spreads  out,  fluctuations 
of  superficial  tension  at  the  edges  of  the  drop  cause  deformations,  and 
result  in  a  species  of  "fraying"  of  the  edges,  minute  particles  of  the  oil 
breaking  off  to  form  a  milky  emulsion  which  gradually  spreads  through 
the  solution. 

In  the  Emulsions  of  oil  in  water  which  are  thus  formed  the  spherical 
droplets  of  oil  are  surrounded  and  completely  enveloped  by  the  water. 
The  power  of  a  given  quantity  of  water  to  surround  oil  must  evidently 


EMULSION-STRUCTURE  OF  PROTOPLASM  287 

be  limited,  however,  for  no  matter  how  tightly  packed  the  particles  of 
oil  may  be,  the  thickness  of  the  layer  of  water  between  them  cannot  be 
of  less  than  molecular  dimensions  at  its  thinnest,  and  must  of  course 
be  much  greater  in  the  interstices  of  the  emulsion.  In  other  words 
a  limited  quantity  of  water  cannot  emulsify  an  unlimited  quantity  of 
oil  and,  as  a  matter  of  fact,  when  a  given  quantity  of  oil  is  shaken  with 
varying  proportions  of  alkaline  water,  if  the  volume  of  water  is  below 
a  certain  critical  fraction  of  the  volume  of  the  oil,  the  character  of  the 
emulsion  which  is  obtained  is  altered  altogether,  and  we  now  have 
emulsions  of  Water  in  Oil.  A  convenient  method  of  symbolically 
representing  these  differing  types  of  emulsions  is  to  enclose  the  Internal 
Phase  of  the  emulsion  in  brackets.  Thus  an  emulsion  of  oil  in  water 
would  be  designated: 

water  (oil) 

while  an  emulsion  of  water  in  oil  would  be  distinguished  by  the  symbol : 

(water)  oil 

When  the  proportion  of  water  to  oil  is  in  the  neighborhood  of  the 
critical  ratio,  complex  intermediate  forms  of  emulsion  may  be  encoun- 
tered, such  as  emulsions  of  oil  in  water  emulsified  in  oil,  thus : 

((oil)  water)  oil 

It  is,  in  fact,  highly  probable  that  the  majority  of  emulsions  of  water 
in  oil  are  really  of  this  more  complex  type. 

The  character  of  an  emulsion  obtained  by  shaking  together  olive 
oil  and  alkaline  water  may  very  readily  be  ascertained  without  micro- 
scopical examination  by  the  simple  device  of  sprinkling  upon  the  surface 
of  the  emulsion  a  little  finely  powdered  Sudan  III  or  Scarlet  R.  These 
dyes  are  soluble  in  oil,  but  insoluble  in  water.  Hence  if  they  are 
sprinkled  upon  the  surface  of  an  emulsion  of  oil  in  water,  the  dye 
simply  dissolves  in  and  stains  the  drops  of  oil  with  which  it  comes  into 
actual  contact,  leaving  the  remainder  of  the  emulsion  unstained.  If, 
however,  the  emulsion  is  one  in  which  water  is  the  internal  phase  and 
oil  the  external,  the  dye  dissolves  in  the  interstitial  oil  and  spreads  over 
the  surface  of  the  emulsion. 

The  following  are  illustrative  results  obtained  by  shaking  together 
olive  oil  and  water- at  an  approximately  uniform  rate  of  shaking,  and 
in  the  presence  of  a  fixed  proportion  of  alkali : 

Components  of  emulsion. 
Oil  Water  5N.NaOH 

c.c.  c.c.  c.c.  Character  of  the  emulsion  obtained. 

(Water)  oil;  fluid,  yellow. 

(Water)  oil;  fluid,  yellow. 

(Water)  oil ;  fluid,  yellow. 

(Water)  oil;  fluid,  creamy. 

(Water)  oil;  fluid,  creamy. 

Water  (oil) ;  white,  very  viscous. 


98  1 

96  3 

92  7 

91  8 

90  9 

89  10 


Water  (oil) ;  white,  very  viscous. 


The  critical  ratio  was  in  this  instance: 


^ 


oil  90.5 


288    PROPERTIES  CONFERRED  BY  COLLOIDAL  CONSTITUENTS 

The  value  of  the  critical  ratio  varies  with  different  samples  of  oil, 
because  of  their  varying  fatty-acid  content.  It  also  varies  with  the 
proportion  of  alkali  employed,  since  if  the  quantity  of  soap  be  insuffi- 
cient to  surround  all  the  droplets  with  a  layer  of  molecular  thickness 
the  stable  emulsification  of  the  whole  of  a  large  excess  of  oil  becomes  an 
impossibility,  and  the  critical  ratio  is  increased. 

It  will  be  observed  that  upon  passing  the  critical  ratio  the  char- 
acteristics of  the  emulsion  change  very  markedly.  Instead  of  the 
yellow,  fluid  emulsions  obtained  while  water  is  the  internal  phase, 
creamy  and  more  viscous  emulsions  result  when  water  is  the  external 
phase.  In  the  neighborhood  of  the  critical  ratio  the  viscosity  of  the 
water  (oil)  emulsions  is  very  greatly  enhanced,  and  emulsions  of  an 
almost  butter-like  consistency  may  be  obtained.  This  probably  arises 
from  the  fact  that  when  the  water  in  the  emulsion  is  just,  and  only  just 
sufficient  to  surround  all  of  the  oil-droplets,  any  deformation  whatever 
of  the  tightly-packed  oil-droplets  must  increase  the  size  of  the  interstices 
between  them;  but  this  can  only  be  accomplished  by  a  complete  disrup- 
tion and  inversion  of  the  emulsion,  since  the  water  is  already  stretched 
to  the  utmost  limit  of  its  covering-power.  The  viscosity,  or  resist- 
ance to  deformation  of  these  emulsions,  therefore,  represents  the  force 
required  to  invert  their  structure. 

Not  only  the  lipoid  constituents  of  cells,  but  also  the  Proteins  tend 
to  form  films  at  the  surfaces  of  suspended  droplets,  and  thus  facilitate 
the  formation  of  emulsions.  If  Chloroform  be  shaken  up  with  pure 
distilled  water  no  emulsion  arises;  the  two  liquids  separating  com- 
pletely after  a  very  brief  interval.  If,  however,  a  protein  be  added  to 
the  water  the  chloroform,  instead  of  separating  out  in  the  form  of  a 
continuous  layer,  separates  out  in  small  discrete  droplets  which,  if 
numerous,  form  a  milky  layer  at  the  bottom  of  the  vessel;  by  trans- 
mitted light,  however,  they  appear  perfectly  transparent.  These 
droplets  are  extraordinarily  stable  and  do  not  coalesce,  however  long 
they  may  stand  in  contact  with  one  another.  They  may  be  repeatedly 
washed  in  water  until  all  traces  of  protein  have  disappeared  from  the 
wash-fluid,  and  they  still  remain  perfectly  stable  and  distinct  from  one 
another.  They  may  be  shaken  up  in  chloroform  itself  or  treated 
with  dilute  sodium  hydroxide  solution  without  impairing  their  form  or 
stability.  If,  however,  they  be  heated  to  nearly  the  boiling-point  of 
chloroform  under  a  layer  of  water  the  droplets  burst  and  coalesce, 
forming  a  homogeneous  layer  of  chloroform.  If  treated  with  alcohol 
they  immediately  dissolve  leaving  a  fine  membranous  precipitate  of 
protein  floating  in  the  water.  Thus  if  we  shake  up  chloroform  with 
about  twice  its  volume  of  a  one  per  cent,  solution  of  Protamine  Sulphate 
or  a  one  per  cent,  solution  of  Gelatin,  and,  after  allowing  the  droplets 
to  settle,  pour  off  the  supernatant  fluid  and  repeatedly  wash  the  drop- 
lets with  water,  then  if  we  suspend  these  droplets  in  a  small  amount  of 
water  and  add  to  the  water  an  equal  volume  of  Alcohol  and  gently 
shake  the  test-tube,  the  droplets  which  are  thus  stirred  up  into  the 


EMULSION-STRUCTURE  OF  PROTOPLASM  289 

'  alcohol-water  layer  can  be  seen  to  swell  up  rapidly  and  burst,  and  the 
fine  membranes  which  surrounded  them  can  then  be  seen  falling 
down  through  the  alcohol-water.  If  we  now  add  several  volumes  of 
alcohol  and  shake  up  the  liquid,  the  chloroform  droplets  all  disappear 
and  what  we  now  have  is  a  clear,  homogeneous  solution,  in  which 
innumerable  minute  membranes  can  be  clearly  seen  floating. 

The  phenomena  of  Relative  Semipermeability  may  also  be  illustrated 
by  these  droplets.  Substances  which  are  soluble  in  water  and  also  in 
chloroform  penetrate  the  membranes,  and  if,  like  Alcohol,  Ether  or 
Ethyl  Acetate  they  chance  to  be  more  soluble  in  chloroform  than  in 
water,  the  chloroform  in  the  droplets  may  take  up  so  much  of  the  sub- 
stance that  they  swell  to  the  extent  of  rupturing  their  enveloping 
membranes.  If,  however,  the  substances  in  which  the  droplets  are 
immersed  are  sufficiently  insoluble  in  water  they  fail  to  penetrate  the 
membranes  and  then  the  droplets  may  be  "  plasmolyzed,"  that  is, 
the  chloroform  may  be  extracted  from  them  leaving  the  enveloping 
membranes  shrunken  and  empty.  This  occurs  when  the  droplets  are 
suspended  in  Toluol,  Xylol  or  Carbon  Bisulphide. 

Fat  emulsions  which  contain  protein  tend  to  form  films  at  surfaces 
with  which  they  come  in  contact,  consisting  of  a  more  concentrated 
emulsion,  both  in  respect  to  fat  and  in  respect  to  protein,  than  that 
which  constitutes  the  body  of  the  liquid.  This  is  very  well  illustrated 
by  the  film  which  forms  on  the  surface  of  Milk  when  it  is  heated.  The 
heating  of  the  milk  renders  the  Calcium  Caseinate  which  it  contains 
somewhat  less  soluble,  and  the  concentrated  layer  of  calcium  caseinate 
and  fat  particles  which  forms  at  the  surface  becomes,  at  temperatures 
above  45°,  sufficiently  viscous  to  assume  the  consistency  of  a  semi-solid 
film,  which,  owing  to  its  high  viscosity,  does  not  readily  pass  back  into 
solution  upon  cooling.  A  pure  solution  of  calcium  caseinate  becomes 
markedly  opalescent  on  heating  to  45°  but  does  not  form  a  sufficiently 
viscous  film  at  its  surface  to  be  mechanically  separable  from  the 
underlying  liquid. 

A  living  cell  consists  essentially  of  a  more  or  less  finely  emulsified 
suspension  of  fat-like  substances  in  a  semi-gelatinous  solution  of  pro- 
tein. The  film  which  forms  at  the  surface  of  warm  milk  may  be 
regarded  as  an  extreme  illustration  of  the  type  of  surface-layer  which 
we  may  therefore  expect  to  exist  at  the  periphery  of  living  cells,  namely 
an  emulsion  of  fat  and  protein,  more  concentrated  and,  therefore, 
more  viscous  than  the  emulsion  which  constitutes  the  underlying 
protoplasm. 

The  emulsion-structure  of  the  superficial  layer  in  cells  enables  us  to 
account  for  a  very  widespread  property  of  living  cells  which  would 
otherwise  be  almost  inexplicable,  namely  the  property  of  One-sided 
Permeability.  This  phenomenon  is  very  well  illustrated  by  the  follow- 
ing experiment  of  Overton's:  If  tadpoles  are  immersed  in  a  five  or 
six  per  cent,  solution  of  cane-sugar  or  a  0.6  per  cent,  solution  of  sodium 
chloride  they  are  unaffected  either  in  size  or  in  any  other  notable 
19 


290    PROPERTIES  CONFERRED  BY  COLLOIDAL  CONSTITUENTS 

respect.  If,  however,  they  are  immersed  in  solutions  which  are 
hypertonic  to  these,  for  example  in  eight  per  cent,  cane-sugar  or  0.8 
per  cent,  sodium  chloride,  they  lose  a  quantity  of  water  and  in  twenty- 
four  hours  they  are  found  to  have  shrunk  decidedly  in  volume.  Evi- 
dently, then,  the  sugar  or  salt  cannot  enter  the  limiting  membranes  of 
the  cells  of  the  skin  while  water  can  pass  through  them  freely  in  the 
direction  tissues  ->  external  medium.  One  might  imagine,  therefore, 
that  the  epithelium  of  a  tadpole  resembles  an  ordinary  semipermeable 
membrane,  permitting  the  passage  of  water  but  not  of  dissolved 
substances.  If  this  were  really  the  case,  then  on  immersing  the  tad- 
poles in  solutions  which  are  hypotonic  to  0.6  sodium  chloride  we  should 
expect  them  to  take  up  water  and  to  increase  in  volume  just  as  much  as 
they  decrease  in  volume  in  hypertonic  solutions.  This  does  not  occur, 
however,  and  tadpoles  immersed  in  hypotonic  solutions  do  not  take  up 
water  to  any  greater  extent  than  from  isotonic  solutions.  We  can  only 
infer,  therefore,  that  the  superficial  epithelium  of  the  tadpole  permits 
the  passage  of  water  from  within  outward,  but  not  in  the  reverse 
direction;  that  this  membrane  is  permeable  to  water  in  the  direction 
tissues  ->  external  medium,  but  not  in  the  direction  external  medium 
->  tissues. 

The  property  of  one-sided  permeability  is  displayed  by  many  living 
membranes,  but  not  by  all.  A  very  striking  contrast  is  shown  in  this 
respect  by  the  Pavement  Epithelium  which  lines  the  peritoneal  cavity, 
on  the  one  hand,  and  the  Columnar  Epithelium  which  lines  the  lumen  of 
the  small  intestine,  on  the  other.  Thus  Heidenhain  introduced  50  c.c. 
of  a  three  per  cent,  solution  of  Glucose  into  the  peritoneal  cavity  of  a 
dog,  and  at  the  same  time  44  c.c.  of  the  same  solution  into  an  isolated 
loop  of  the  intestine.  After  ninety  minutes  the  quantity  and  composi- 
tion of  the  residual  fluid  in  the  peritoneal  cavity  were  as  follows : 

Quantity  of  fluid.  Glucose.  Sodium  chloride. 

19.5  c.c.  1 .  0  per  cent.  0 . 55  per  cent. 

while  in  twenty-five  minutes  the  composition  of  the  residual  fluid  in 
the  loop  of  intestine  was  as  follows : 

Quantity  of  fluid.  Glucose.  Sodium  chloride. 

19 . 0  c.c.  3 . 8  per  cent.  0 .  04  per  cent. 

From  the  peritoneal  cavity  both  water  and  glucose  had  issued  into 
the  tissue-fluids,  the  glucose  even  more  rapidly  than  the  water,  while 
sodium  chloride,  which  was  absent  from  the  fluid  originally  introduced, 
had  diffused  from  the  tissues  into  the  peritoneal  cavity.  The  peri- 
toneal epithelium,  therefore,  behaved  like  a  membrane  of  parchment, 
permitting  the  passage  of  dissolved  substances  in  either  direction  in 
proportion  to  their  relative  concentrations  on  the  two  sides  of  the 
membrane. 

From  the  intestinal  loop,  both  water  and  glucose  had  issued  into  the 
tissue-fluids,  water  somewhat  more  rapidly  than  glucose.  But  prac- 
tically no  sodium  chloride  had  diffused  into  the  intestinal  fluid  from  the 


EMULSION-STRUCTURE  OF  PROTOPLASM  291 

tissue-fluids.  Evidently  the  intestinal  epithelium  permits  the  passage 
of  certain  dissolved  substances  into  the  tissue-fluids  behind  it,  but  not 
the  migration  of  dissolved  substances  in  the  reverse  direction. 

The  maintenance  of  one-sided  permeability  in  tissues  is  dependent 
upon  the  maintenance  of  the  unimpaired  structure  of  the  cells.  Thus 
the  phenomenon  of  one-sided  permeability  is  nowhere  more  clearly 
illustrated  than  it  is  in  the  kidneys,  where  the  dissolved  constituents 
of  Urine  are  constantly  excreted  against  a  high  pressure,  the  tissues  of 
the  kidney  being  much  more  permeable  for  dissolved  substances  in  the 
direction  blood  -» urine  than  in  the  direction  urine  -»  blood.  If,  how- 
ever, the  epithelium  of  the  renal  tubules  is  injured  by  perfusion  with 
solutions  of  certain  substances,  for  example  Sodium  Fluoride,  it  loses 
this  power  and  comes  to  resemble  much  more  closely  a  membrane  of 
parchment.  This  is  very  clearly  illustrated  by  the  following  experi- 
ment by  Bottazzi.  One  kidney  in  a  dog  was  injured  by  perfusion  with 
sodium  fluoride  solution.  The  ureters  of  the  two  kidneys  were  then 
separately  catheterized  and  the  freezing-points  of  the  samples  of  urine 
collected  from  the  two  kidneys  were  determined  from  time  to  time. 
The  following  were  illustrative  results : 

Depression  of  Depression  of 

Urine  c.c.:                         freezing-point.  freezing-point 

Time.                                                     • ..        . • of  blood. 

Normal.         Injured.          Normal.          Injured. 


2.30  to  3.00 

3.30  to  4.00 

4.50  to  5.20 

5. 30  to  6.00 

6.00  to  6.30 

9.30  to  10.00 

8.00  to  8.30 


9.  12  1.616  0.979               0.572 

14.  20  1.118  0.294 

14.  22  0.584  0.240 

10.  22  0.570  0.224 

12.  20  0.572  0.212               0.560 

4.  9  1.002  0.206 

2.5  6  1.304  0.302               0.569 


It  is  evident  that  the  phenomenon  of  one-sided  permeability  must  be 
dependent  upon  a  heterogeneous  structure  of  the  membrane  which 
displays  it.  The  phenomenon  is  not,  and  could  not  be  displayed  by 
structureless  membranes,  or  by  membranes  having  a  uniform  structure 
in  the  direction  of  penetration,  i.  e.,  perpendicularly  to  their  surface. 
For  instance,  consider  a  membrane  formed  of  successive  columns,  of 
two  different  materials,  one  of  which  permits  the  passage  of  substances 
soluble  in  water  while  the  other  does  not.  Then  if  the  arrangement 
of  these  two  components  were  th,at  displayed  in  Fig.  15  substances 
soluble  in  water  could  penetrate  the  unshaded  channels  just  as  easily 
from  below  as  from  above  the  membrane.  But  if  the  membrane  were 
curved,  so  as  to  bring  the  columns  of  impenetrable  material  closer 
together  on  the  under  than  on  the  upper  surface,  or  if  they  were 
pyramidal  in  shape  so  as  to  achieve  the  same  end,  so  that  the  arrange- 
ment would  be  that  displayed  in  Fig.  16  then  it  is  evident  that  the 
penetrable  area  on  the  under  surface  of  the  membrane  would  be  a 
much  smaller  proportion  of  the  whole  area  than  on  the  upper  surface 
of  the  membrane,  so  that  substances  penetrating  from  above  would 
do  so  with  comparative  ease,  while  substances  issuing  from  below  the 
membrane  would  do  so  with  difficulty. 


292     PROPERTIES  CONFERRED  BY  COLLOIDAL  CONSTITUENTS 

Now  the  lipoid  constituents  of  the  cell  may  be  assumed  to  be  gener- 
ally impenetrable  by  substances  which  are  insoluble  in  fats  and  oils, 
so  that  these  must  seek  entry  to  the  cell  through  the  interstitial  spaces 
between  the  lipoid  constituents  of  the  superficial  membrane  of  the  cell. 
If,  therefore,  these  intersitital  spaces  filled  with  a  solution  or  gel  of 
protein,  were  so  constructed  as  to  be  narrower  at  one  end  than  at  the 
other,  the  superficial  membrane  of  the  cell  would  evidently  be  more 
readily  permeable  in  one  direction  than  in  the  opposite. 

The  most  usual  spatial  arrangement  of  the  various  structures  or 
constituents  of  a  cell  is  that  of  Radial  Symmetry.  The  primitive 
arrangement  of  strictly  radial  symmetry  so  frequently  displayed  in 
spherical  cells  becomes  modified  or  distorted  in  those  cells,  such  as  the 
majority  of  epithelial  cells,  which,  through  mutual  compression  or  for 
other  reasons,  have  assumed  a  columnar,  stratified  or  flattened  outline. 
In  such  cases  the  radial  arrangement  of  structures  may  be  confined  to 
the  sides  or  margins  of  the  cell,  and  differ  in  character  in  the  protoplasm 
underlying  the  various  facets  of  the  cell. 


FIG.  15  FIG.  16 

FIGS.  15  and  16. — These  figures  illustrate  the  effect  of  curvature  of  the  surface  of  a 
radially  dispersed  emulsion  in  producing  funnel-shaped  interstitial  pores  between  the 
radiating  columns  of  lipoid  globules.  In  Fig.  15  both  surfaces  of  the  cortical  layer 
being  plane,  the  diameter  of  the  interstitial  orifices  are  the  same  at  the  exterior  and 
interior  surfaces.  In  Fig.  16  the  cortical  layer  being  curved,  the  interstitial  orifices 
are  narrower  upon  the  un  der  than  upon  the  upper  surface  of  the  cortical  layer. 

A  radial  arrangement  of  the  ultramicroscopic  fat-granules  of  the  cell 
would  obviously  lead  to  the  formation  upon  the  surface  and  in  the 
subjacent  protoplasm  of  minute  Funnel-shaped  Pores,  of  which  the 
interstitial  openings  would  be  permeable  to  substances  soluble  in  water, 
while  the  walls,  being  composed  of  fat-granules,  would  be  impermeable 
or  with  difficulty  permeable  by  such  substances.  The  interstitial 
openings  at  the  margin  distal  from  the  center,  from  which  the  fat- 
granules  radiate,  would  be  relatively  large,  while  at  points  lying  nearer 
to  the  center  of  radiation,  that  is,  in  general,  deeper  within  the  cell, 
the  diameter  of  the  pores  would  be  very  considerably  contracted. 

Substances  which  are  soluble  in  water  might  evidently  penetrate 
such  a  cell  with  relative  ease,  since  a  relatively  large  proportion  of  the 
exterior  cell-surface  would  consist  of  the  water-soluble  phase  of  the 
emulsion,  but  they  would  issue  from  the  interior  of  the  cell  with 
relative  difficulty,  since  a  relatively  large  proportion  of  the  area  which 
they  would  have  to  traverse  to  find  an  outlet  would  consist  of  the 
lipoidal  phase.  If  the  modifications  of  radial  symmetry  which  are 
so  characteristic  of  Epithelial  Cells  should  result  in  the  confinement  of 
this  structure  to  one  surface  or  facet  of  the  cell,  it  is  obviously  possible 
that  one-sided  permeability  of  a  tissue  composed  of  such  cells  might 


EMULSION-STRUCTURE  OF  PROTOPLASM  293 

be  the  consequence.  This  may  be  seen  by  reference  to  the  diagram 
in  Fig.  16. 

It  should  be  borne  in  mind  that  the  existence  of  funnel-shaped  pores 
in  the  surface  of  a  cell  or  in  a  membrane  would  only  give  rise  to  one- 
sided permeability  provided  the  diameter  of  the  pore  at  the  constricted 
end  were  comparable  with  the  mean  free  path  of  the  penetrating  mole- 
cules. Were  the  least  diameter  of  the  pores  less  than  the  mean  free 
path  of  the  pentrating  molecule,  then  the  membrane  would  be  a  strictly 
semipermeable  membrane  for  this  type  of  molecule.  Were  the  least 
diameter  of  the  pores  on  the  contrary,  very  large  in  comparison  with 
the  mean  free  path  of  the  molecule  concerned,  then  the  membrane 
would  be  freely  permeable  by  this  molecule  in  either  direction.  Thus 
it  is  readily  conceivable  that  membranes  of  this  type  might  exhibit 
One-sided  Permeability  for  certain  substances  dissolved  in  water, 
Absolute  Permeability  for  others,  and  Semipermeability  for  yet  other 
molecules. 

On  the  other  hand,  if  the  above  sketch  represents  truly  the  structure 
of  the  superficial  layer  of  cells,  substances  which  are  soluble  in  fats 
would  enter  the  cell  through  the  radiating  columns  of  lipoidal  material. 
Now  the  phenomenon  of  one-sided  permeability  has  not  as  yet  been 
observed  to  be  displayed  toward  substances  which  are  soluble  in 
lipoids.  Indeed,  in  general,  the  penetrability  of  cells  by  substances 
which  are  soluble  in  lipoids  is  very  much  greater  than  their  penetra- 
bility by  other  substances,  no  matter  how  soluble  they  may  be  in  water. 
This  fact  is  very  strikingly  illustrated  by  the  experiments  of  Overton 
and  Meyer,  who  measured  the  minimal  concentrations  of  various 
Narcotics  dissolved  in  water  which  would  induce  narcosis  in  tadpoles, 
the  narcosis  being  evidenced  by  cessation  of  movement.  The  same 
narcotics  were  dissolved  in  water  and  the  water  shaken  up  in  Olive 
Oil  and  the  relative  solubilities  of  the  narcotics  in  water  and  in  oil 
estimated  by  the  distribution  of  the  narcotic  between  the  two  solvents. 
The  following  were  illustrative  results  obtained  with  various  Alcohols  : 


Solubility  in  water. 

Narcotic.  molecules  per  liter.  Solubility  in  oil. 

Methyl  alcohol  .      .      .      .      0.52-0.62  Solubility  in  water  =  OC      solublein50 

parts  of  oil. 

Ethyl  alcohol     ....      0.27-0.31  30     :      1 

Propyl  alcohol    ....      0.11  8:1 

Butyl  alcohol     ....      0.038  Soluble   in    12   parts  of     solubility  in 

water;  oil  =  OC  . 

Caprylic  alcohol       .      .      .      0.0004  Soluble  in  2000  parts  of     solubility  in 

water;  oil  =  (X  . 

The  following  results  were  obtained  with  other  narcotics: 

'    Solubility  in  oil. 

Critical  concentration.  Solubility  in  water. 

Narcotic.                           .  ------  •  -  •  •  --  •  -    -  • 

At  3°.            At  30°.  At  3°.               At  30°. 

Salicylamide       ....     Vwoo             i/600  22.232              14.002 

Benzamide    .....     l/5oo              Yaoo  0.672               0.437 

Monacetin    .....     V»o               l/io  0.099               0.066 

Ethyl  alcohol      ..*..»/«                 V»  0-026                0.047 

Chloral  hydrate       .      .      .     VBO               V*o  0.053               0.236 

Acetone  ......     J/3                 VT  0.146               0.235 


294    PROPERTIES  CONFERRED  BY  COLLOIDAL  CONSTITUENTS 

Hence  if  tadpoles,  anesthetized  at  30°  by  a  2f0  solution  of  Chloral 
Hydrate  be  cooled  they  recover  their  mobility,  on  warming  they  are 
again  anesthetized,  and  at  30°  the  solubility  of  chloral  hydrate  in  olive 
oil  is  much  greater  than  it  is  at  lower  temperatures. 

While  the  inference  drawn  by  Overton  and  Meyer  from  these  experi- 
ments, that  only  those  substances  which  are  soluble  in  Lipoids  can 
penetrate  the  cell,  obviously  cannot  be  substantiated,  for  otherwise 
neither  water,  inorganic  salts  nor  amino-acids  could  ever  gain  entry  into 
protoplasm,  yet  it  is  very  manifest  from  these  and  many  other  experi- 
ments of  a  like  nature  that  substances  which  are  soluble  in  lipoids  do 
enter  living  cells  with  exceptional  ease.  We  may  probably  infer  with 
safety  that  the  lipoidal  elements  in  the  superficial  membranes  of  cells 
constitute  a  large  proportion  of  the  surface,  and  the  interstices  a  rela- 
tively small  proportion,  so  that  substances  which  are  insoluble  in  lipoids 
enter  living  cells  with  comparative  difficulty 


FIG.  17. — Illustrating  the  increase  in  the  diameter  of  the  interstitial  spaces  which  results 
from  increase  in  the  diameter  of  the  fat-droplets  in  an  emulsion. 

Any  reagent  or  condition  which  affects  the  State  of  Aggregation 
of  the  fat-globules  in  the  limiting  membrane  of  cells  must  necessarily 
affect  the  diameter  of  the  interstices  between  them.  In  general  those 
conditions  involving  the  formation  of  large  aggregates  would  increase 
the  permeability  of  tissue  by  enlarging  the  diameter  of  the  radiating 
fat-droplets  and,  therefore,  that  of  the  interstitial  spaces  (Fig.  17). 
This  is  very  strikingly  illustrated  by  the  eggs  of  certain  marine  forms 
such  as  the  sea-urchin  which,  when  exposed  to  the  action  of  fat-solvents 
become  permeable  to  water  which  they  take  up  from  the  surrounding 
sea-water.  The  water  thus  absorbed  accumulates  in  a  layer  just  under 
the  superficial  membrane  of  the  cell,  lifting  it  off  the  underlying  pro- 
toplasm and  forming  the  "Fertilization  Membrane"  which  is  normally 
the  effect  of  a  cytolytic  agent  carried  into  the  egg-cell  by  the  head 
of  the  spermatozoon.  TJie  permeability  of  the  surface  of  the  cell  is 
also  increased  for  inorganic  salts,  for  McClendon  has  shown  that  the 
Electrical  Conductivity  of  a  suspension  of  sea-urchin  eggs  is  increased 
by  fertilization  while  Osterhout  has  shown  that  an  increase  in  the 
electrical  conductivity  of  living  tissues  is  indicative  of  increased  per- 
meability of  the  surface  of  the  cell  for  inorganic  salts. 

Since  the  lipoidal  droplets  in  cells  are  suspended  in  a  gelatinous  or 


VISCOSITY  OF  PROTOPLASM  295 

semi-liquid  solution  of  Protein  we  may  also  assume  that  any  condition 
tending  to  alter  the  consistency  of  the  interstitial  protein  solution  would 
deform  the  structure  of  the  emulsion.  Coagulating  Agents  especially 
might  be  expected  to  reduce  the  interstitial  gel  to  discrete  granules  or 
separate  flocculi,  thus  removing  the  obstacle  to  coalescence  of  the  fat 
globules  and  the  consequent  coarsening  of  structure  and  widening  of 
interstices.  Corresponding  to  this  conception  we  find  that  simple 
heating  of  a  piece  of  frog's  skin  renders  it  freely  permeable  to  water 
in  either  direction,  instead  of  only  in  one.  The  effect  of  coagulating 
agents'  upon  permeability  may  also  be  strikingly  illustrated  in  the 
following  way:  If  paramcecia  be  washed  free  from  culture  medium 
with  pure  distilled  water  and  suspended  in  a  solution  of  Methyl  Green 
(free  from  methyl  violet),  the  protoplasm  of  the  infusorians  takes  on  a 
faint  greenish  tinge,  but  the  large  pseudo-nucleus  remains  white  and 
unstained.  After  removing  the  excess  of  methyl  green  by  washing  the 
organisms  in  distilled  water,  a  little  Cupric  Chloride  may  now  be  added 
to  the  water.  Immediately  the  nucleus  becomes  stained  a  deep  green, 
indicating  that  the  impenetrability  of  the  nuclear  membrane  for  the 
dye  prevents  it  from  being  stained  in  the  normal  cell,  but  after  the 
action  of  this  protein  coagulant,  which  kills  the  organisms,  the  permea- 
bility of  the  nuclear  membrane  is  so  enhanced  that  the  dye  can  readily 
enter  and  even  attain  a  greater  concentration  therein  than  it  does  in 
the  cytoplasm. 

THE  VISCOSITY  OF  PROTOPLASM. 

The  major  part  of  the  high  degree  of  Viscosity  which  protoplasm 
displays  is  attributable  to  the  Protein  which  it  contains.  The  viscosity 
of  a  protein  solution  increases  very  rapidly  indeed  with  its  concentra- 
tion, so  rapidly,  in  fact,  that  earlier  observers  were  inclined  to  the 
belief  that  the  viscosity  changed  suddenly  at  definite  critical  con- 
centrations instead  of  changing  evenly  and  with  regularity  as  it  does 
in  solutions  of  crystalloids.  Later  observations  have  shown  us,  how- 
ever, that  the  viscosity  of  protein  solutions  increases  with  the  concen- 
tration in  accordance  with  the  usual  formula  ~0  =  An,  where  77  is  the 

viscosity  of  the  solutiorr,  170  that  of  the  solvent,  n  the  concentration  of 
the  solution  and  A  a  constant,  the  numerical  value  of  which  depends 
upon  the  nature  of  the  dissolved  substance,  and  upon  the  temperature. 
The  following  are  results  obtained  by  Sackur,  employing  Sodium 
Caseinate : 

n  (in  equivalents  -  (15°  C.).  logio  A. 

of  sodium) .  % 

0.01830  1.870  14.8 

0.01370  1.581  14.5 

0.00915 1.363  14.3 

0.00547 1.202  14.6 

0.00458  ,  1.165  14.5 


296     PROPERTIES  CONFERRED  BY  COLLOIDAL  CONSTITUENTS 

A  remarkable  feature  of  these  results  is  the  extraordinarily  high 
value  of  A,  involving  a  very  rapid  increase  of  viscosity  with  increasing 
concentration.  For  the  majority  of  crystalloids  the  value  of  A  is  not 
greatly  in  excess  of  unity,  while  for  sodium  caseinate  it  is  of  the  Order 
of  1014.  This  fact  alone  would  lead  us  to  suspect  that  the  mechanism 
which  produces  the  viscosity  of  these  solutions  is  different  in  nature 
from  that  which  produces  the  viscosity  of  solutions  of  crystalloids. 
The  viscosity  of  a  protein  solution  is  also  very  greatly  increased  by 
lonization,  the  viscosity  of  protein  solutions  being  at  a  minimum  when 
ionic  protein  is  absent,  i.  e.,  when  the  protein  is  uncombined  with  acids 
or  bases. 

Indeed  a  very  little  consideration  suffices  to  show  that  the  viscosity 
of  a  protein  solution  is  of  a  very  different  type  from  the  viscosity,  for 
example,  of  solutions  of  Sugar  or  Glycerol  in  water.  Apart  from  the 
extraordinary  magnitude  of  A,  the  type  of  viscosity  exhibited 
by  solutions  of  proteins  differs  from  the  viscosity  of  a  glycerol-water 
mixture  in  that  it  affords  no  hindrance,  or  very  slight  hindrance, 
to  the  motion  of  ions  and  of  crystalloidal  molecules.  The  velocities 
with  which  various  crystalloids  diffuse  through  Gelatin  jellies  are 
remarkably  close  to  the  diffusion-velocities  of  the  same  substances  in 
distilled  water.  The  jelly  causes  a  very  slight  retardation  of  diffusion 
but  the  hindrance  to  molecular  movement  is  disproportionately  small 
in  comparison  with  the  enormous  viscosity  of  the  jellies. 

It  has  repeatedly  been  shown  that  the  specific  mobilities  of  the 
majority  of  inorganic  ions  is  the  same  in  gelatin  or  agar  jellies  as  it  is 
in  distilled  water.  In  fact,  if  allowance  be  made  for  the  diminution 
of  the  cross-section  of  the  conducting  field  which  is  occasioned  by  the 
presence  of  gelatin  molecules  we  find  that  the  electrical  conductivities 
of  inorganic  salt  solutions  in  gelatin  jellies  are  only  very  slightly  less 
than  those  of  equally  concentrated  solutions  in  pure  water,  implying 
that  the  ions  of  the  electrolyte  move  as  freely  in  the  insterstices  between 
the  protein  molecules  as  they  would  move  in  distilled  water.  This  is 
true  even  when  the  ions  are  protein  ions,  for  the  dependence  of  the 
Electrical  Conductivity  of  protein  solutions  upon  their  dilution  is  of  a 
perfectly  normal  character,  resembling  the  dependence  of  the  conduc- 
tivity of  a  solution  of  a  crystalloid  upon  dilution,  although,  in  the  range 
of  concentrations  employed,  the  viscosity  of  the  protein  solution 
increases  with  its  concentration  very  greatly,  while  that  of  a  salt 
solution,  for  example,  increases  almost  imperceptibly. 

On  the  other  hand  the  intimate  dependence  of  the  conductivities 
of  solutions  of  electrolytes  upon  the  ordinary  types  of  viscosity  has 
been  commented  upon,  and  quantitatively  estimated  by  a  host  of 
observers.  Viscosities,  very  much  less  than  those  of  the  most  dilute 
Jellies,  if  caused  by  such  substances  as  sugar  or  glycerol,  profoundly 
diminish  the  conductive  power  of  electrolytes.  Not  only  inorganic,  but 
also  protein  ions  are  very  greatly  hindered  in  their  mobilities  by  the 
type  of  viscousness  which  alcohol-water  or  glycerol-water  mixtures 


VISCOSITY  OF  PROTOPLASM  297 

exhibit.  In  fact,  whereas  doubling  the  viscosity  of  a  solution  of 
Sodium  Caseinate  by  the  addition  of  protein  does  not  measurably  affect 
its  conductivity,  doubling  its  viscosity  by  the  addition  of  forty  per  cent, 
of  alcohol  reduces  the  mobility  of  the  caseinate  ions  to  one-half,  and  the 
conductivity  of  the  solution  to  a  still  smaller  proportion.  In  estimating 
the  influence  of  viscosity  upon  the  mobilities  of  protein  ions  we  can 
entirely  disregard  that  portion  of  the  viscosity  of  the  solution  which, 
although  comparable  in  magnitude  with  the  viscosity  of  the  solvent, 
is  attributable  to  the  protein  itself. 

There  are  thus  two  kinds  of  viscosity  which  may  be  displayed  by 
solutions,  the  one  which  impedes  the  motion  of  molecules  or  ions,  and 
the  other  which  does  not  hinder  the  motion  of  such  small  particles, 
although  it  does  very  greatly  impede  the  passage  of  the  fluid  through 
a  narrow  tube  or  the  rate  of  oscillation  of  a  rotating  disc  suspended 
within  the  fluid.  The  former  type  of  viscosity  is  displayed  by  solutions 
of  inorganic  substances  and  the  simpler  organic  substances,  the  latter 
type  of  viscosity  by  solutions  of  the  proteins. 

The  customary  method  of  measuring  viscosity,  such  as  the  measure- 
ment of  the  time  taken  by  a  given  volume  of  fluid  to  pass,  under  the 
force  of  gravity,  through  a  specified  length  of  a  narrow  tube,  all  involve 
deformation  of  the  fluid,  whereas  the  estimation  of  viscosity  which 
depends  upon  the  diffusion  of  molecules  or  ions  through  it,  does  not 
require  any  displacement  of  the  particles  of  the  solvent  in  which  the 
diffusion  is  occurring.  Deformation  is  especially  resisted  by  protein 
solutions,  but  internal  molecular  motions  are  not  impeded.  This  fact 
strongly  suggests  the  existence  of  a  Structure  within  solutions  of  the 
proteins.  It  appears  very  probable  that  the  molecules  of  protein  in 
solution  are  loosely  connected  with  one  another  so  as  to  form  a  mesh- 
work  or  three-dimensional  net  throughout  the  body  of  the  solution. 
Such  a  net,  which,  in  two-dimensional  section,  we  may  picture  as  some- 
thing analogous  to  a  tennis-net  with  microscopic  or  ultramicroscopic 
meshes,  would  offer  no  hindrance  to  the  passage  through  it  of  a  quickly- 
moving  body  which  is  much  smaller  than  its  meshes,  but  to  any  force 
involving  deformation  of  its  structure,  for  instance  to  a  force  tending 
to  drag  it  through  a  small  tube,  it  would  offer  a  very  considerable 
resistance.  In  measuring  the  resistance  which  a  protein  solution 
offers  to  passage  through  a  capillary  tube,  we  are  not  measuring  true 
viscosity  or  internal  friction  between  adjacent  molecules,  therefore, 
but  the  resistance  of  the  structure  of  the  solution  to  deformation. 

A  common  method  of  measuring  the  viscosity  of  fluid  consists  in 
suspending  a  disc  within  the  fluid  and  causing  it  to  oscillate,  the  decrease 
of  the  rate  of  oscillation  being  a  measure  of  the  viscosity. 

When  this  method  is  applied  to  protein  solutions,  however,  it  is 
found  that  the  decrease  in  the  rate  of  oscillation  of  the  disc  is  abnormally 
rapid,  but  if  the  liquid  be  slightly  shaken  or  the  disc  taken  out  and 
replaced,  the  decrease  in  the  rate  of  oscillation  becomes  normal  again 
for  a  brief  period.  Evidently  the  protein  network  adheres  to  the  disc, 


298    PROPERTIES  CONFERRED  BY  COLLOIDAL  CONSTITUENTS 

so  that,  in  the  course  of  time,  the  motion  of  the  disc  is  not  merely 
opposed  by  the  friction  of  immediately  adjacent  molecules,  but  by  the 
inertia  of  all  of  the  protein  molecules  of  the  fluid  which  are  attached 
indirectly,  through  a  continuous  meshwork,  to  the  oscillating  disc. 

JELLIES  AND  GELATINIZATION. 

The  structure  which  confers  upon  protein  solutions  their  peculiar 
type  of  Viscosity  leads  in  many  cases  when  the  solutions  are  sufficiently 
concentrated  to  their  acquiring  certain  of  the  properties  of  solids. 
Such  solutions  are  what  we  term  Jellies,  and  they  resemble  solids  in 
presenting  pronounced  resistance  to  deformation  which,  however, 
yields  to  the  slightest  force  if  its  action  be  sufficiently  prolonged. 
Where  forces  of  an  instantaneous  character  are  concerned,  therefore, 
the  jellies  are  solids,  but  where  forces  of  prolonged  action  are  concerned 
they  are  fluids.  The  distinction  between  a  solid  and  a  jelly  is,  in  fact, 
largely  a  matter  of  degree.  A  solid  will  flow  under  a  sufficiently  great 
pressure  applied  for  a  relatively  brief  period  of  time,  but  a  sharp  impact 
affects  it  as  it  affects  an  elastic  solid,  causing  oscillation  and  recoil, 
but  not  deformation.  Intermediate  states  of  matter  are  afforded  by 
such  materials  as  Sealing-wax  which,  even  at  ordinary  temperatures, 
will  flow  under  slight  pressure  applied  for  very  prolonged  periods, 
but  which  under  even  considerable  forces  acting  sufficiently  suddenly 
exhibits  all  the  brittleness  of  a  solid. 

Under  certain  conditions,  when  the  meshes  are  sufficiently  coarse, 
various  jellies  or  "gels"  clearly  display  a  network  or  spongy  structure. 
If  an  insoluble  gel,  such  as  White  of  Egg  coagulated  by  fixatives,  the  gel 
of  Colloidion  produced  by  the  action  of  chloroform  upon  an  ether 
solution,  common  black  India-rubber,  or  the  hydrogel  of  Silica  be 
examined  under  high  magnification  they  can  all  be  seen  to  possess  a 
fine  sponge-like  structure.  When,  for  example,  a  thirteen  per  cent, 
solution  of  egg-white  is  fixed  with  sublimate,  sections  are  found  to 
show  a  sponge- structure,  or,  what  corresponds  to  a  sponge  in  two 
dimensions,  a  network-structure.  W.  B.  Hardy,  who  has  especially 
investigated  this  gel,  failed  to  obtain  with  acid  or  basic  dyes  any 
staining  of  the  substance  within  the  meshes  of  the  net,  and  pressure 
applied  to  the  gel  resulted  in  the  squeezing  of  fluid  out  of  its  interstices. 
The  structure  of  the  gel  is,  therefore,  that  of  an  open  sponge-work  of 
solid,  containing  fluid  within  its  meshes.  Direct  experimentation  with 
A  gar  jellies  has  shown  that  in  a  gel  containing  one  r>er  cent,  of  agar,  the 
solid  framework  is  a  solution  of  water  in  agar,  while  the  fluid  in  the 
interstices  is  a  dilute  solution  of  agar  in  water.  Upon  heating  the 
solution  the  two  components  become  miscible  in  each  other  and  we 
obtain  what  appears  to  be  a  homogeneous  solution.  Upon  the  basis 
of  these  facts  Hardy  draws  a  far-reaching  analogy  between  the  jellies 
which  liquefy  when  heated,  and  gel  when  cooled,  and  the  system 
Phenol-water,  which,  if  it  contains  more  than  71  per  cent,  or  less  than 


JELLIES  AND  GELATINIZATION  299 

76  per  cent,  of  phenol,  separates,  at  temperatures  below  80°  C.,  into 
two  phases,  the  one  a  solution  of  phenol  in  water,  the  other  a  solution  of 
water  in  phenol.  According  to  the  view  developed  by  Hardy  the  two 
cases  differ  only  in  the  fact  that  upon  separation  of  the  two  phases  in 
the  agar-water  system  the  system  retains  a  structure,  while  in  the 
phenol-water  system  no  structure  is  retained  and  the  components 
separate  into  two  clearly  demarcated  layers.  Essentially,  the  dif- 
ference between  the  two  systems  consists  in  this :  that  when  the  phenol- 
water  system  separates  into  two  phases,  the  phases  become  separated 
by  the  minimal  possible  surface,  namely  a  plane;  while  when  the  agar- 
water  system  separates  into  two  phases  they  remain  in  contact  over  an 
area  far  larger  than  the  minimum.  In  the  latter  case  it  would  seem 
that  the  surface-tension  at  the  interface  of  the  two  phases  is  very  low, 
so  that  the  force  leading  to  the  diminution  of  surface  is  small.  The 
resistance  to  the  diminution  of  the  interface  is  also  very  large  because 
of  the  high  viscosity  of  the  gel. 

The  manner  in  which  the  structure  of  a  gel  is  built  up  can  be  readily 
observed  in  the  ternary  mixture,  alcohol,  gelatin  and  water.  If  13.5 
grams  of  Gelatin  are  mixed  with  50  c.c.  of  water  and  50  c.c.  of  absolute 
alcohol,  a  mixture  is  formed  which  is  optically  homogeneous  at  17°  to 
20°  C.,  but  which  separates  into  two  phases  at  temperatures  below 
this.  Hardy  thus  describes  the  sequence  of  events  on  cooling  this 
mixture  below  the  temperature  of  gelation:  "As  the  temperature  falls 
below  the  limit  a  clouding  occurs  which  I  find  to  be  due  to  the  appear- 
ance of  fluid  droplets  which  gradually  increase  in  size  until  they 
measure  3  /z.ju.  On  cooling  further,  these  fluid  droplets  become 
solid  and  they  begin  to  adhere  to  one  another.  The  framework  is 
therefore  an  open  structure  which  holds  the  fluid  phase  in  its  inter- 
stices." "When  once  formed  the  phases  have  considerable  stability. 
If  the  droplets  are  composed  of  a  solid  solution  one  may,  by  the  addi- 
tion of  water,  cause  them  to  increase  to  relatively  vast  dimensions 
without  their  being  destroyed;  as  they  increase  in  size  their  refractive 
index  approximates  more  and  more  to  that  of  the  external  phase  until 
they  are  finally  lost  sight  of.  The  addition  of  alcohol,  however,  once 
more  brings  them  into  view  and  causes  them  to  shrink.  Owing  to 
this  stability,  once  a  configuration  has  been  established,  one  has  to  far 
overstep  the  conditions  of  its  formation  in  order  to  destroy  it.  This 
would  account  for  the  remarkable  hysteresis  observed  in  reversible 
gels.  .  .  .  When  water  is  added  to  a  ternary  mixture  so  as  to 
considerably  swell  the  droplets,  the  system  is  unstable,  and  the  two 
phases  mix  at  once  when  it  is  mechanically  agitated." 

In  jellies  of  this  type  which  are  dilute  with  respect  to  the  colloid 
constituent,  therefore,  the  structure  is  that  of  an  open  sponge-work, 
the  meshes  being  filled  with  water  or  a  water-rich  solution  of  the 
substance  forming  the  gel  while  the  framework  of  the  sponge  consists 
of  anastomosing  threads  composed  of  linearly  arranged  globules  of  the 
water-poor  phase.  In  such  gels,  therefore,  the  surface  of  the  water- 


300     PROPERTIES  CONFERRED  BY  COLLOIDAL  CONSTITUENTS 

rich  phase  is  concave;  in  other  words  the  water-proof  phase  is  the 
Internal  Phase  of  the  gel,  and  the  water-rich  material  constitutes  the 
External  Phase  of  the  gel.  If,  however,  to  a  ternary  mixture  of  gelatin, 
alcohol  and  water  which  forms  such  a  gel  as  that  described  above, 
more  gelatin  be  added,  the  character  of  the  gel  changes  entirely  and 
its  structure  becomes  inverted.  The  water-poor  phase  becomes  con- 
cave and  the  water-rich  phase,  instead  of  being,  as  formerly,  concave, 
becomes  convex  to  it.  On  cooling  such  a  mixture  to  a  temperature 
below  that  at  which  it  forms  an  optically  homogeneous  solution, 
droplets  separate  out  which  are  poor  in  gelatin,  while  the  interstitial 
portion  of  the  system,  which  is  rich  in  gelatin,  solidifies.  Thus  the 
gel  comes  to  possess  a  Honeycomb-structure  the  droplets  being  poor  in 
gelatin  and  rich  in  water.  This  is  very  clearly  shown  by  the  following 
analyses  made  by  Hardy : 

TEMPERATURE  OF  THE  MIXTURE,  15°  C.  (EQUAL  PARTS  OF  WATER  AND 

ALCOHOL). 


Per  cent, 
gelatin  in 
mixture. 
67     

Per  cent,  gelatin  in 
droplets  (internal 
phase)  . 
17.0 

Per  cent,  gelatin  in 
interstices  (external 
phase). 
2.0 

13  5     

.      .      .      .      .      .      18.0 

5  5 

36.5     , 

8.5 

40.0 

From  these  analyses  it  is  also  clear  that  the  two  phases  in  a  protein 
gel  are  not  of  constant  composition,  but  may,  under  different  conditions 
of  total  concentration,  etc.,  vary  widely  in  their  relative  and  absolute 
gelatin  and  water-content.  This  system  differs,  therefore,  from  the 
system  phenol-water,  not  only  in  the  extent  of  the  surface  which 
separates  the  phase,  but  also  in  the  variability  of  the  composition  of 
its  phases,  in  this  respect  resembling  rather  the  system  hydrated 
silica-water. 

The  reason  for  this  inversion  of  structure  which  occurs  in  concen- 
trated gelatin  Jellies  is  the  same  as  that  which  is  the  origin  of  the 
inversion  of  structure  in  olive-oil-water  Emulsions  when  the  proportion 
of  oil  to  water  is  increased  beyond  a  certain  limit.  The  spreading-  or 
covering-power  of  water  is  not  unlimited  and  therefore  the  amount  of 
oil  or  gelatin  which  it  can  surround  is  correspondingly  restricted. 

The  question  has  been  raised  whether  the  jelly  which  is  formed  by 
gelatin  dissolved  in  water  (instead  of  alcohol-water  mixtures)  really 
possesses  a  structure  analogous  to  that  observed  by  Hardy  in  ternary 
systems.  It  has  been  urged  that  this  structure  is  an  artefact  arising  out 
of  partial  Coagulation  of  the  protein,  since  it  is  not  directly  visible  in 
binary  systems.  The  action  of  coagulants  such  as  alcohol  or  sublimate 
upon  jellies  which  already  possess  a  structure  of  this  type,  however,  is 
not  to  otherwise  alter,  but  merely  to  coarsen  their  structure.  This  is  due 
to  loss  of  water  on  the  part  of  the  colloid-rich  droplets  with  a  consequent 
diminution  of  the  volume  of  the  colloid-rich  phase  and  an  increase  in 
the  volume  of  the  more  fluid  interstices.  This  can  be  shown,  not  only 


JELLIES  AND  GELATINIZATION  301 

by  direct  observation,  but  also  by  the  relative  ease  with  which  water 
can  be  expressed  from  the  jelly  before  and  after  "fixation."  From 
Poiseuilles'  Law  for  the  outflow  of  liquid  from  capillary  tubes,  it  follows 
that  the  pressure  required  to  express  the  fluid  must  vary  approximately 
as  the  inverse  fourth  power  of  the  diameter  of  the  meshes,  although, 
of  course,  the  variable  viscosity  of  the  expressed  fluid  will  be  a  factor 
introducing  departures  from  this  simple  law.  Now  a  hydrogel  con- 
taining 13  per  cent,  of  Gelatin  at  a  temperature  of  15°  C.  will  endure  a 
pressure  of  400  pounds  to  the  square  inch  without  expression  of  water; 
after  fixation  with  formalin  or  corrosive  sublimate,  however,  the  fluid 
can  be  expressed  from  the  gel  like  water  from  a  sponge,  with  simple 
hand-pressure. 

Since  more  complete  coagulation  does  not  alter  the  type  of  structure 
possessed  by  jellies  of  partially  coagulated  protein,  but  merely  coarsens 
it,  it  is  a  fair  inference  that  jellies  which  have  undergone  no  measure 
of  coagulation  also  possess  the  type  of  structure  outlined  by  Hardy, 
but  that  owing  to  its  fineness  the  details  of  this  structure  are  not 
visible. 

The  existence  of  a  structure  in  jellies  formed  by  the  solution  of  gelatin 
in  water  is  also  objectively  demonstrated  by  the  observation  of  Liese- 
gang,  that  when  silver  nitrate  diffuses  into  gelatin  which  is  impregnated 
with  potassium  bichromate,  the  precipitation  of  insoluble  Silver 
Bichromate  does  not  occur  indifferently  in  all  parts  of  the  area  of  diffu- 
sion, but  in  concentric  circles.  It  has  also  been  shown  by  Rohonyi 
that  when  thin  films  of  gelatin  are  frozen  the  ice-crystals  are  formed 
in  concentric  rings.  It  is  difficult  to  clearly  conceive  any  mechanism 
which  would  permit  this  in  a  perfectly  homogeneous  medium.  The 
theory  that  crystallization  is  inhibited  by  the  gelatin  until  a  certain 
degree  of  supersaturation  is  attained  might  account  for  failure  of 
precipitation  or  crystallization  at  certain  points,  but,  provided  the 
jelly  were  strictly  homogeneous  and  structureless,  it  fails  to  account  for 
its  appearance  at  other  points. 

The  experiments  of  Hardy  show  that  on  adding  water  to  the  system 
alcohol-water-gelatin  the  gelatin-rich  phase  progressively  imbibes 
water  until  it  passes  by  a  series  of  insensible  transitions  into  a  Solution 
of  gelatin.  We  have  seen  that  solutions  of  protein  show  evidence,  in 
the  peculiar  type  of  resistance  to  deformation  which  they  display,  of 
possessing  a  structure  which  is  most  easily  conceived  as  a  spongework 
of  protein  molecules  with  intercommunicating  meshes  filled  with  water. 
The  Structure  of  the  solution  is  therefore  that  of  an  attenuated  jelly 
and  there  is  no  distinction  of  kind,  but  only  of  degree,  between  a 
protein  solution  and  a  protein  jelly.  As  a  matter  of  fact,  if  the  Viscosity 
of  a  solution  of  gelatin  sufficiently  concentrated  to  gelatinize  at  room- 
temperature  be  measured  at  intervals  while  it  is  cooling,  no  sharp 
change  of  viscosity  is  found  to  occur  at  gelation,  the  viscosity  of  the 
solution  just  prior  to  that  point  being  so  great  as  to  afford  clear  indica- 
tion of  the  forthcoming  semi-solidification. 

The  structure  of  protoplasm,  therefore,  consisting  as  it  does  of  an 


302    PROPERTIES  CONFERRED  BY  COLLOIDAL  CONSTITUENTS 

emulsion  of  lipoids  suspended  in  a  solution  or  jelly  of  protein  must  be 
very  complex.  Essentially  it  is  an  emulsion  enclosed  within  another 
emulsion  and  many  diversities  of  structure  and  arrangement  may 
evidently  exist.  One  would  anticipate  that  the  architecture  of  such  a 
complex  system  would  be  profoundly  affected  by  any  factors  affecting 
the  solubility  of  the  proteins,  and  therefore  their  affinity  for  water. 
A  relative  alteration  of  volume  of  the  water-poor  and  water-rich  phases 
of  the  protein  emulsion  must  necessarily  disturb  all  the  space-relations 
of  the  enclosed  fat-emulsion,  and  these  displacements  acting  at  the 
Surface  of  the  cell  would  be  equivalent  to  opening  or  shutting  so  many 
doors  for  the  entry  of  water-soluble  substances  into  the  cell.  The 
striking  effects  of  various  inorganic  substances  upon  the  Permeability 
of  cells  upon  which  we  shall  dwell  in  the  succeeding  chapter,  probably 
originate  in  changes  of  the  affinity  of  the  cell-proteins  for  water  with 
consequent  dilatations  or  contraction  of  the  constituent  phases  of  the 
protein  jelly  and  enlargement  or  constriction  of  the  interstitial  spaces 
between  the  lipoidal  elements  of  the  superficies  of  the  cell. 

THE  OSMOTIC  PRESSURE  OF  PROTEIN  SOLUTIONS. 

It  was  formerly  believed  that  proteins  in  solution  exerted,  in  common 
with  other  colloids,  either  no  osmotic  pressure  at  all,  or  a  pressure  of 
immeasurably  small  extent.  More  recent  investigations  have  shown, 
however,  that  the  difference  in  this  as  in  other  respects  between  the 
colloids  and  the  "typical"  crystalloids  is  merely  a  quantitative  differ- 
ence which  is  directly  attributable  to  and  deducible  from  the  relatively 
enormous  size  of  their  molecules.  Thus  a  one  per  cent,  solution  of 
Glucose  contains  y-g-  gram-molecules  of  glucose  per  liter  and  exerts 
an  osmotic  pressure  of  nearly  one  and  a  quarter  atmospheres,  but  a 
one  per  cent,  solution  of  Hemoglobin,  which  has  a  molecular  weight  of 
sixteen  thousand,  only  contains  y^ro  gram-molecules  of  protein  per 
liter  and,  therefore,  may  be  expected  only  to  exert  an  osmotic  pressure 
of  0.014  of  an  atmosphere. 

The  direct  determination  of  the  osmotic  pressure  of  protein  solutions 
is  a  task  fraught  with  immense  difficulties,  on  account  of  the  difficulty 
of  preparing  ideally  pure  proteins.  The  investigations  of  Graham, 
the  originator  of  the  distinction  between  crystalloids  and  colloids, 
appeared  to  indicate  that  colloids  in  general  exert  a  high  osmotic 
pressure.  Subsequent  investigators,  however,  attributed  these  results 
to  an  admixture  of  crystalloids,  which,  as  the  above  numerical  compari- 
son shows,  might  be  expected  to  exert  a  disproportionate  effect  upon 
the  pressures  exhibited.  Starling  endeavored  to  measure  directly  the 
osmotic  pressure  of  the  proteins  in  blood-serum  by  using  for  his  Osmom- 
eter  a  membrane  permeable  to  salts  but  impermeable  to  proteins,  and 
this  method  has,  since  then,  been  employed  in  all  accurate  work  upon 
the  subject,  since,  as  Reid  has  pointed  out,  it  is  the  only  method  of 
procedure  which  is  applicable  to  the  problem.  Such  a  membrane  is 
to  the  colloids  what  an  ideally  semipermeable  membrane  is  to  all 


OSMOTIC  PRESSURE  OF  PROTEIN  SOLUTIONS  303 

dissolved  substances,  inclusive  of  the  colloids.  We  have  no  assurance 
that  any  given  protein  preparation  is  totally  free  from  impurities  which 
may  influence  the  direct  measurement  of  osmotic  pressure;  it  is,  there- 
fore, essential  to  employ  a  membrane  which  is  permeable  to  such  im- 
purities and  thus,  if  time  be  allowed  for  the  system  to  come  to  equi- 
librium, differentiates  between  protein  and  non-protein  constituents 
of  the  solution  under  investigation.  For  this  purpose  Reid  employs 
a  membrane  of  vegetable  parchment,  which,  as  he  has  shown,  is  per- 
meable even  to  nucleic  acid,  although  it  is  impermeable  to  the  proteins 
which  he  employed  in  his  investigations.  By  extremely  prolonged 
purification  Reid  has  succeeded  in  obtaining  preparations  of  Egg- 
albumin  which  exhibit  no  measurable  osmotic  pressure  when  examined 
by  this  method.  In  subsequent  investigations,  however,  he  obtained 
osmotic  pressures,  due  to  dissolved  Hemoglobin  of  perfectly  constant 
value  and  such  as  to  indicate  a  molecular  weight  of  about  48,000. 
Barcroft  and  Hill  have,  however,  demonstrated  by  thermodynamical 
methods  that  in  solutions  containing  hemoglobin  prepared  by  less 
prolonged  dialysis  the  molecular  weight  of  this  substance  is  close  to 
16,669  which  is  the  figure  calculated  from  the  content  of  Iron,  assuming 
each  molecule  of  hemoglobin  to  contain  only  one  atom  of  iron.  Roaf, 
employing  the  differential  osmotic  method  just  described,  finds  that  the 
molecular  weight  of  hemoglobin,  dissolved  in  distilled  water,  is  about 
32,000,  while  in  sodium  carbonate  solutions  it  is  16,000.  These  results 
appear  to  show  that  when  protein  is  uncombined  with  acids  or  bases 
it  is  polymerized,  and  so  exerts  a  considerably  smaller  pressure  than 
protein  salts. 

The  extremely  important  discovery  has  been  made  by  R.  S.  Lillie, 
that  the  osmotic  pressure  which  is  exerted  by  proteins  (determined 
differentially  as  described)  varies  very  pronouncedly  with  the  nature 
of  the  inorganic  acids  bases  or  salts  which  their  solutions  contain.  The 
following  are  illustrative  results,  obtained  when  dilute  acids  or  alkalies 
are  employed  as  solvents: 

1.5  PER  CENT.  GELATIN  IN  DILUTE  HC1  SOLUTIONS. 

Osmotic  pressure  of 


Solve 
Water 

W/3100  H 
-/2050 
W/1550 
W/1025 
TO/770 
m/620 
™/412 

the  protein  in 
nt.                                                                                                                 mm.  Hg 

.  -  'i                         ,                                 82 

21      ..                                                                                            68 

12.3 
'  '      a     .      .      .  -  •  .   •  +•    ...     .     V    .                                          17  9 

*.»....,.....                                    26  5 

.      32  4 

34.9 
39.3 

1.5  PER  CENT.  GELATIN  IN  DILUTE   KOH  SOLUTIONS. 

Osmotic  pressure  of 

the  protein  in 
Solvent.  mm.  Hg 

Water 7.9 

"YsiooKOH 14.1 

m/62o        " 23.7 

m/412  " 25.1 

m/no        "       ....      ?      ...      r      ..      ?      ..,.     29. Q 


304    PROPERTIES  CONFERRED  BY  COLLOIDAL  CONSTITUENTS 

In  Lillie's  words,  "  In  the  presence  of  either  acid  or  alkali  the  osmotic 
pressure  of  the  gelatin  thus  shows  a  marked  increase,  which,  within  the 
above  range  of  concentrations,  exhibits  a  certain  proportionality  to  the 
quantity  of  acid  or  alkali  added.  For  equivalent  concentrations  acid 
produces  a  somewhat  greater  increase  than  alkali.  The  change  in 
osmotic  properties  is  to  be  attributed  to  a  finer  subdivision  of  the  col- 
loidal particles  and  a  consequent  increase  in  the, surface  of  intersec- 
tion between  colloidal  particles  and  medium."  The  osmotic  pressure  of 
gelatin  and  of  egg-albumin  is  unaffected  by  the  addition  of  non-electro- 
lytes, such  as  cane-sugar,  glucose,  glycerol  and  urea,  but  is  considerably 
affected  by  the  addition  of  Inorganic  Salts,  being  depressed  thereby. 
The  decrease  of  the  osmotic  pressure  exerted  by  the  protein  depends 
upon  the  nature  of  both  the  anion  and  the  cation  of  the  added  salt. 
The  depression  increases  in  the  order:  Alkali  metals  <  alkaline 
earths < heavy  metals  (for  cations);  and  CNS<I<Br<NO3<Cl<F< 
plurivalent  anions,  SCX,  tartrate,  citrate,  phosphate  (for  anions). 
This  fact  is  especially  significant  when  we  recollect  that  this  is  the 
order  in  which  the  various  ions  bring  about  the  dehydration  and  coagu- 
lation of  proteins  (see  Chapter  VIII). 

THE  SWELLING  OF  PROTEIN  JELLIES. 

The  proteins,  as  we  have  seen,  exert  a  small,  but  a  definite  osmotic 
pressure.  They  are  at  the  same  time  not  diffusible  through  jellies 
or  only  very  slightly  so.  Any  crystalloid  which  may  chance  to  be 
present  in  the  external  fluid  which  bathes  a  Gelatin  plate,  therefore, 
can  penetrate  the  gelatin,  although  perhaps  more  slowly  than  water. 
The  gelatin  from  the  interior  of  the  plate  cannot  similarly  escape  into 
the  surrounding  medium.  The  gelatin  plate,  therefore,  acts  like  an 
osmometer  which  provides  its  own  membrane  which  is  permeable  for 
water  and  crystalloids  but  not  for  colloids.  Hence,  when  dry  gelatin 
or  concentrated  jellies  are  placed  in  water  they  take  up  water  and 
increase  in  volume. 

A  phenomenon  in  the  domain  of  crystalloids  which  presents  some 
analogies  to  the  swelling  of  colloidal  jellies  is  the  following :  If  we  place 
at  the  bottom  of  a  column  of  distilled  water  a  layer  of  Phenol  and  intro- 
duce below  this  a  layer  of  saturated  solution  of  potassium  chloride  in 
water  and  now  allow  the  system  to  stand  at  constant  temperature,  the 
layer  of  phenol  gradually  moves  up  the  column  of  water,  in  other 
words  the  layer  of  solution  below  the  phenol  "swells."  The  solvent, 
water,  being  soluble  in  phenol,  the  phenol  is  permeable  by  it,  while  the 
potassium  chloride,  being  insoluble  in  phenol,  cannot  pass  through  the 
supernatant  layer  of  phenol. 

Not  only  osmotic,  but  also  chemical  phenomena  must,  however, 
play  a  part  in  the  swelling  of  protein  jellies.  As  we  have  seen  in 
Chapter  VIII  the  passage  of  a  protein  into  solution  involves  the  addi- 
tion of  the  elements  of  water  to  terminal  — NH2  and  — COOH  groups 


SWELLING  OF  PROTEIN  JELLIES  305 

and  also,  possibly,  to  internal  — N.HOC —  -groups,  resulting  in  the 
Depolymerization  of  the  protein.  Not  only  osmotic  phenomena,  but 
Hydration  of  the  gelatin  must,  therefore,  occur  in  the  process  of  swelling. 
Confirmation  of  this  view  is  afforded  by  the  fact  that  the  Swelling  of 
gelatin  is  accompanied  by  an  absorption  of  heat.  Evidently  the 
processes  of  solution  and  swelling  are  each  composed  of  two  factors, 
one  leading  to  a  disengagement  and  the  other  to  the  absorption  of 
heat.  The  former  process  is  the  chemical  binding  of  water  by  the 
protein,  the  latter  the  passage  of  the  hydrated  protein  into  solution 
(or,  in  swelling,  the  osmotic  imbibition  of  water).  In  swelling,  the 
chemical  heat-effect  predominates,  in  the  dissolving  of  the  gelatin, 
the  heat-effect  of  solution. 

The  degree  of  swelling  which  Gelatin  plates  undergo  in  water  is 
greatly  enhanced  by  the  addition  of  small  amounts  of  acid  or  alkali  to 
the  water,  the  minimal  imbibition  of  water  being  at  a  reaction  very 
close  to  the  neutral  point.  The  phenomena  attending  the  swelling  of 
plates  of  gelatin  in  acidified  water  have  recently  been  very  thoroughly 
investigated  by  Procter.  This  investigator  has  found  that  gelatin 
absorbs  both  acid  and  water  from  acid  solutions,  but  absorbs  the  acid 
in  excess,  so  that  the  proportion  of  acid  in  the  surrounding  fluid  dimin- 
ishes. If  the  initial  concentration  of  acid  in  the  external  fluid  lies 
between  0.01  and  0.25  N,  then,  assuming  that  at  the  end  of  the  process 
(attainment  of  maximal  swelling)  the  concentration  of  free  acid  is  the 
same  within  and  without  the  jelly,  having  been  equalized  in  the  course 
of  time  by  diffusion,  the  amount  of  acid  which  is  "bound"  by  the 
gelatin  is  0.7  to  0.8XlO~3  (=70  to  80XlO~5)  equivalents  per  gram. 
The  equivalence  at  the  attainment  of  maximal  swelling  is  the  same  for 
all  strong  acids,  but  falls  below  this  value  for  weak  acids.  While  the 
proportion  of  acid  which  is  "bound"  by  the  gelatin  varies  but  slightly 
with  the  concentration  of  the  acid  in  the  surrounding  fluid,  this  is  not 
true  of  the  degree  of  swelling  attained,  which  in  strongly  acid  solutions 
attains  its  maximum  at  a  dilution  below  that  required  for  complete 
fixation  of  the  acid  by  the  gelatin,  and  then  falls  in  a  continuous  curve 
with  increasing  concentration  of  the  external  acid  solution.  The  same 
inhibition  of  swelling  is  brought  about  in  varying  degree  by  strong 
solutions  of  various  inorganic  salts,  and  is  attributable  to  the  dehydra- 
tion of  the  protein  which,  in  still  stronger  solutions,  culminates  in  its 
Coagulation. 

The  taking  up  of  water  by  gelatin  from  acid  solutions  is  accounted 
for  by  Procter  in  the  following  way:  He  pictures  the  gelatin  acid- 
compound  as  a  coherent  mass  from  which  the  gelatin  molecules  cannot 
diffuse  or  separate  and  which,  in  most  respects,  behaves  like  a  single 
enormous  complex  molecule.  It  is  reasonable,  he  considers,  to  visualize 
it  as  a  felted  mass  of  amino-acid  chains  held  to  each  other  by  attrac- 
tions which  possibly  attach  only  their  ends,  but  freely  admitting  the 
passage  of  liquid  between  them.  He  assumes,  in  accordance  with  our 
older  conception  of  the  mode  of  formation  and  ionization  of  protein 
20 


306    PROPERTIES  CONFERRED  BY  COLLOIDAL  CONSTITUENTS 

salts,  that  the  compound  yields  acid  anions  (for  example,  chlorine  ions 
when  the  compound  is  gelatin  hydrochloride),  but  that  these  anions, 
although  diffusible,  are  held  within  the  mass  by  the  electrostatic  attrac- 
tion of  the  oppositely  charged  ion  which,  being  colloidal,  cannot  leave 
the  jelly  in  the  company  of  its  associate.  The  only  way,  therefore,  in 
which  the  osmotic  pressure  of  the  anions  can  take  effect  is  not  by  their 
own  movement,  but  by  the  inward  movement  of  water,  resulting  in 
the  swelling  of  the  entire  jelly-mass  and  its  dilution  by  admixture  with 
the  outside  solution. 

Two  very  serious  objections  attach  to  this  interpretation  of  the 
phenomena.  In  the  first  place,  as  Procter  himself  has  pointed  out, 
were  this  the  actual  mechanism  of  swelling,  then  the  operative  force 
compelling  movement  of  the  water  would,  in  ultimate  analysis,  be  the 
Electrostatic  Tension  which  prevents  the  issuance  of  the  inorganic 
anions  from  the  jelly  into  the  solution.  There  should  thus  be  a 
measureable  difference  of  electrical  potential  between  the  swollen 
gelatin  jelly  and  the  surrounding  medium.  Such  a  difference  of  poten- 
tial has  not  been  found.1  In  the  second  place,  as  Procter  also  points 
out,  another  difficulty  lies  in  the  fact  that  the  condition  which  would 
thus  arise,  would  offer  no  equilibrium,  since  the  concentration  of  the 
inorganic  anions  and  the  free  acid  itself  could  never  become  simul- 
taneously equal  within  and  without  the  jelly;  no  matter  what  degree  of 
swelling  and  consequent  dilution  of  the  jelly  may  have  occurred  there 
will  still,  ex  hypothesi,  be  an  excess  of  inorganic  anions  within  the  jelly. 

Our  more  recent  views  regarding  the  mode  of  formation  and  ionization 
of  Protein  Salts  reconcile  both  these  difficulties,  however,  for,  since  we 
now  know  that  no  inorganic  ions,  or  at  most  a  very  small  proportion, 
are  yielded  by  the  protein  acid  compound,  the  swelling  of  the  jelly 
must  be  due,  just  as  it  is  in  the  case  of  gelatin  immersed  in  neutral  water, 
to  the  osmotic  pressure  of  the  colloidal  particles  themselves,  which,  being 
unable  to  penetrate  the  colloidal  network  in  which  they  are  entangled, 
necessarily  compel  the  compensating  migration  of  water.  No  electro- 
static tension  between  the  jelly  and  the  external  solution  need  be 
assumed,  because  both  ionic  components  of  the  protein  salt  are  attract- 
ing water  by  virtue  of  their  osmotic  pressure,  and  they  are  necessarily 
present  in  the  jelly  in  equimolecular  concentration  since  neither  of 
them  can  leave  it,  even  in  the  minimal  quantity  necessary  to  create 
an  electrostatic  tension.  Furthermore,  since  no  inorganic  anions  are 
yielded  by  the  protein  salt,  simultaneous  equality  of  concentration 
of  the  uncombined  acid  and  the  acid  anions  within  and  without  the 
jelly  will  be  simply  assured  by  their  equal  and  unhampered  diffusion 
into  the  jelly.  The  increased  Swelling-capacity  of  gelatin  in  solutions 
of  acids  or  alkalies  is  merely  the  expression  of  the  fact  that  the  ionization 

1  Ehrenberg,  R.:  Biochem.  Ztschr.,  1913,  53,  p.  356.  Even  if  both  of  the  ions 
of  the  protein  salt  were  able  to  issue  from  the  jelly,  a  difference  of  potential  would  arise 
from  their  unequal  speeds  of  diffusion.  See  F.  E.  Bartell  and  C.  D.  Hocker:  Jour. 
Am.  Chem.  Soc.,  1916,  38,  pp.  1029  and  1036. 


SWELLING  OF  PROTEIN  JELLIES  307 

of  the  protein  salt  leads  to  an  increase  in  the  number  of  colloidal  par- 
ticles per  unit  volume  of  the  jelly,  and  possibly,  also,,  in  part,  to  the 
fact  that  protein  ions  have  a  greater  affinity  for  water  than  undisso- 
ciated  protein  molecules. 

This  conception  of  the  process  of  swelling  would  still  yield  no  equi- 
librium or  Swelling-maximum  were  there  no  compensating  force  acting 
in  an  opposite  sense  to  the  osmotic  pressure  of  the  gelatin  itself.  No 
matter  how  much  gelatin  may  be  diluted  by  swelling,  there  will  always 
remain  an  excess  of  osmotic  pressure  within  the  jelly,  due  to  the  protein 
ions  which  cannot  leave  it.  Now  gelatin  plates,  when  immersed  in 
water  or  in  acid  solutions,  do  not  swell  indefinitely  until  swelling 
merges  insensibly  into  solution,  but,  on  the  contrary,  display  a  definite 
swelling-maximum.  At  this  point,  therefore,  the  osmotic  pressure 
exerted  by  the  colloidal  particles  within  the  jelly  must  be  balanced  by 
an  equal  opposing  force,  which  Procter  interprets  as  the  tension  of 
the  elastic  colloidal  network. 

The  effects  of  Inorganic  Salts  upon  the  swelling  of  gelatin  plates  are 
complex  because,  as  Loeb  has  recently  demonstrated,  they  consist  of 
two  separate  factors:  In  the  first  place  a  chemical  interaction  occurs 
between  the  gelatin  and  the  salt,  leading  to  the  formation  of  a  compound 
of  the  acid  component  of  the  salt  with  the  gelatin.  This  compound 
has  a  greater  swelling-capacity  than  uncombined  gelatin.  On  the 
other  hand  the  uncombined  portion  of  the  salt,  when  present  in  excess, 
tends  in  varying  degree,  depending  upon  the  particular  salt  employed, 
to  dehydrate  the  gelatin  and  therefore  to  inhibit  swelling.  The  power 
of  the  various  salts  to  inhibit  the  swelling  of  gelatin  is  proportionate 
to  their  power  of  coagulating  proteins  in  solution. 

The  swelling  of  Living  Tissues  when  immersed  in  hypotonic  solutions 
or  in  acid  or  alkaline  isotonic  solutions  is  a  very  complex  phenomenon . 
In  the  first  place  it  is  determined  by  the  Permeability  of  the  surfaces 
of  the  cells  for  water  and  anything  affecting  the  permeability  of  the 
cells  of  the  tissue  will  also  influence  the  imbibition  of  water.  In  the 
second  place  the  swelling  of  the  tissue  is  determined  by  the  Osmotic 
Pressure  of  the  proteins  which  it  contains,  which  is  affected  by  acids, 
alkalies  and  salts  in  the  manner  outlined  above  for  gelatin.  In  the 
third  place  degenerative  changes  in  excised  tissues  such  as  the  excised 
muscles  of  the  frog's  leg,  lead  sooner  or  later  to  production  of  diffusible 
products  of  Autolysis,  and  since  the  surface-layers  of  the  tissue  are 
with  difficulty  penetrable  by  some  of  these,  their  production  leads  to  a 
greatly  enhanced  imbibition  of  water. 

Finally,  the  water  which  is  taken  up  by  the  tissue  may  actually 
enter  the  cells  or,  on  the  contrary,  may  merely  be  taken  up  into  the 
interstitial  spaces  between  the  cells.  In  general  it  may  be  stated, 
however,  that  any  factor  tending  to  injure  the  vitality  of  the  cells, 
for  example  heating,  will  greatly  increase  their  permeability  for  water, 
and  hence  will  increase  the  rate  and  degree  of  swelling  in  hypotonic 
solutions. 


308    PROPERTIES  CONFERRED  BY  COLLOIDAL  CONSTITUENTS 

The  fact  that  acids  greatly  increase  the  swelling-capacity  of  Gelatin 
or  Fibrin  has  led  M.  H.  Fischer  to  attempt  to  account  in  this  way  for 
the  edematous  conditions  of  tissues  which  are  encountered  in  a  variety 
of  pathological  conditions.  He  is  of  the  opinion  that  the  Edema  of 
tissues  is  due  to  local  development  of  acids  which  increase  the  affinity 
of  the  tissue-proteins  for  water.  Many  objections  to  this  view  have, 
however,  been  advanced  by  a  number  of  investigators  and  it  does 
not  appear  feasible  to  account  for  the  phenomena  of  edema  in  any 
such  simple  manner.  In  the  first  place  the  buffer-action  of  the  tissues 
and  tissue-fluids  must  undoubtedly  prevent  the  development  of  a 
sufficiently  high  acidity  to  account  for  the  accumulations  of  fluid 
which  occur  in  edema.  The  acidity  required  to  influence  in  so  decided 
a  manner  the  swelling  of  gelatin  or  fibrin,  is  far  greater  than  the  acidity 
which  could  possibly  prevail  within  living  tissues  or  the  tissue-fluids 
derived  from  them,  and  as  a  matter  of  fact  very  considerable  edema 
may  prevail  in  tissues  displaying  no  perceptible  deviation  from  the 
normal  neutral  or  excessively  faintly  alkaline  reaction  of  all  living 
tissues  and  tissue-fluids.  Then,  again,  the  accumulations  of  fluid 
which  occur  in  and  characterize  edema  are  more  frequently  interstitial 
than  intercellular.  The  fluid  is  found  between  the  cells  and  not  within 
the  cells  themselves,  where  the  proteins  are  present  in  highest  concen- 
tration. It  appears  more  probable  that  in  the  majority  of  the  instances 
of  edema,  fluid  accumulates  in  abnormal  situations  because  the  per- 
meability of  the  membranes  lining  the  lymph-spaces  or  finer  blood- 
vessels has  been  increased  by  injury.  Thus  we  know  that  various 
substances  such  as  leech-extract  or  extracts  of  shell-fish  or  peptones,  or 
other  injurious  agencies  such  as  heating,  will  so  greatly  modify  the 
permeability  of  the  capillary  bloodvessels  as  to  lead  to  great  accumula- 
tions of  fluid  in  the  lymph-spaces.  Similar  changes  in  these  or  other 
membranous  surfaces  may  very  probably  account  for  the  accumulation 
of  fluids  in  the  cellular  interstices  of  tissues  in  certain  disease-conditions. 
Even  when  edema  is  accompanied  by  the  accumulation  of  fluid  within 
the  cells  themselves,  this  is  rather  to  be  attributed  to  alterations  of 
permeability  or  of  the  affinity  of  the  proteins  for  water  by  disturbance 
of  the  normal  balance  of  the  inorganic  salts  in  the  protoplasm,  than  to 
local  development  of  acidity. 

REFERENCES. 
GENERAL: 

Hoeber:     Physikalische  Chemie  der  Zelle  und  der  Gewebe.      Leipzig,  4th  edition. 
Robertson:     The  Physical  Chemistry  of  the  Proteins.     New  York,  1918. 
EMULSIONS  AND  SURFACE-TENSION: 

Quineke:     Pflliger's  Arch.,   1879,  19,  p.    129.     Drude's  Annalen,   1901,  7,  p.   631; 

1902,  9,  p.  969;  1903,  10,  p.  507. 
Billschli:     Untersuchungen  iiber  mikroskopische  Schaume  und  das  Protoplasma. 

Leipzig,  1892. 
Ramsden:     Arch.  f.  Anat.  u.  Physiol.,  Physiol.  Abt.,  1894,  p.  517.     Zeit.  f.  physik. 

Chemie,  1904,  47,  p.  336. 

Hardy,  W.  B.:     Jour.  Physiol,  1899,  24,  p.  158. 
Rona  and  Michaelis:     Biochem.  Zeit.,  1907,  5,  p.  365. 


SWELLING  OF  PROTEIN  JELLIES  309 

Robertson:     Zeit.  f.  Chem.  und  Ind.  der  Kolloide,   1908,  2,  p.  49;   1910,  7,  p.  7; 
Arch,  di  Fisiol.,  1909,  7,  p.  189. 

Shorter:     Phil.  Mag.,  6  series,  1909,  17,  p.  560. 

Macallum:     Ergeb.  d.  Physiol.,  1911,  11,  p.  598. 

Bancroft:     Jour.  Physical  Chem.,  1912,  16,  pp.  177,  345,  475,  739;  1913,  17,  p.  501; 

1915,  19,  p.  275;  1916,  20,  p.  1. 
SURFACE-LAYER  OF  CELLS: 

Overton:     Studien  iiber  die  Narkose,  Jena,  1901. 

Nathanson:     Jahrb.  f.  wissenschaftl.  Botan.,  1904,  39,  p.  607. 

von  KnajlH-Lenz:     Pfliiger's  Arch.,  1908,  123,  p.  279. 

Robertson:     Jour.  Biol.  Chem.,  1908,  4,  p.  1.     Science  N.  S.,  1917,  45,  p.  273. 

McClendon:     Am.  Jour.  Physiol.,  1910-11,  27,  p.  240. 

Harvey:     Jour.  Exp.  Zool.,  1910,  8,  p.  355. 

Lillie:     Jour.  Morph.,  1911,  22,  p.  695.     Am.  Jour.  Physiol.,  1910-11,  27,  p.  289. 

Loeb:     Artificial  Parthenogenesis  and  Fertilization,  Chicago,  1913. 
VISCOSITY  : 

Sackur:     Zeit.  f.  physik.  Chem.,  1902,  41,  p.  672. 

von  Schroeder:     Ibid.,  1903,  45,  p.  75. 

Hardy:     Jour.  Physiol.,  1905,  33,  p.  251. 

Dumanski:     Zeit.  f.  physik.  Chem.,  1907,  60,  p.  553. 

Robertson:     Jour.  Physical  Chem.,  1911,  15,  p.  387. 
JELLIES  AND  GELATINIZATION: 

Hardy:     Jour.  Physiol.,  1899,  24,  p.  158.     Jour.  Physical  Chem.,  1900,  4,  p.  254. 
Proc.  Roy.  Soc.,  London,   1899,  66,  p.  110. 

Liesegang:     Zeit.  f.  Chem.  und  Ind.  der  Kolloide,  1907,  1,  p.  364. 

Rohonyi:     Biochem.  Zeit.,  1913,  53,  p.  210. 
OSMOTIC  PRESSURE  OF  PROTEIN  SOLUTIONS: 

Reid:     Jour.  Physiol.,  1904,  31,  p.  438;  1905-6,  33,  p.  12. 

Moore  and  Roaf:     Biochem.  Jour.,  1917,  2,  p.  34;  1908,  3,  p.  55. 

Lillie:     Am.  Jour.  Physiol.,  1907-8,  20,  p.  127. 

Adamson  and  Roaf:     Biochem.  Jour.,  1908,  3,  p.  422. 

Roaf:     Jour.  Physiol.  Proc.,  1909,  38,  p.  1.     Quart.  Jour.  Expt.  Physiol.,  1910,  3, 
pp.  75,  171. 

Moore  and  Bigland:     Biochem.  Jour.,  1911,  5,  p.  32. 

Barcroft  and  Hill:     Jour.  Physiol.,  1910,  39,  p.  411. 
SWELLING  OF  PROTEIN  JELLIES: 

Wiedermann  and  Ludeking:     Ann.  d.  Physik,  N.F.,  1885,  25,  p.  145. 

Hofmeister:     Arch.  f.  exp.  Path.  u.  Pharm.,  1890,  27,  p.  395;  1891,  28,  p.  210. 

Pauli  (Pascheles):     Ibid.,  1895,  36,  p.  100.     Pfliiger's  Arch.,  1897,  67,  p.  219;  1898, 
71,  p.  333. 

Procter:     Trans.  Chem.  Soc.  London,  1914,  105,  p.  313.     Collegium,  1915. 

Procter  and  Burton:     Jour.  Soc.  Chem.  Ind.,  1916,  35,  p.  404. 

Procter  and  Wilson:     Trans.  Chem.  Soc.,  London,  1916,  109,  p.  307. 

Katz:     Zeit.  f.  Physiol.  Chem.,  1916,  96,  p.  255. 

Loeb:     Jour.  Biol.  Chem.,  1917,  31,  p.  343;  1918,  33,  p.  531;  1918,  34,  pp.  77,  395, 
489;  1918,  35,  p.  497.     Jour.  Gen.  Physiol.,  1918-19,  1,  pp.  39,  237,  363  and  483. 
SWELLING  OF  TISSUES: 

Loeb:    Pfliiger's  Arch.,  1898,  69,  p.  1;    1898,  71,  p.  457;  1899,  75,  p.  303.     Am. 
Jour.  Physiol.,  1899-1900,  3,  p.  327. 

Cooke:     Jour.  Physiol.,  1898-99,  23,  p.  137. 

Fischer:     (Edema.     New  York,  1910. 

Moore:     Jour.  Am.  Med.  Assn.,  1912,  59,  p.  423. 

Beutner:     Biochem.  Zeit.,  1912,  39,  p.  230;  1913,  48,  p.  217. 

von  Korosy:     Zeit.  Physiol.  Chem.,  1915,  93,  p.  154. 


CHAPTER  XIV. 

PROPERTIES  CONFERRED  BY  THE  COLLOIDAL  CON- 
STITUENTS; CHEMICAL  AND  BIOLOGICAL. 

EFFECTS  OF  DISTURBANCE  OF  THE  INORGANIC  ENVIRONMENT. 

We  have  already  had  occasion  to  discuss  the  effects  of  alteration  of 
the  total  Concentration  of  the  Environment  upon  the  protoplasm  inhabit- 
ing it,  and  we  have  seen  that  the  most  salient  of  these  effects  depend 
primarily  upon  the  migration  of  water  into  or  out  of  the  substance  of 
the  cell.  The  effects  arising  out  of  alteration  of  the  normal  Compo- 
sition of  the  Environment  appear  to  have  been  first  systematically 
investigated  by  James  Blake,  a  physician  resident  in  San  Francisco 
in  the  decades  comprised  between  the  years  1870  and  1890.  His 
earliest  investigations  upon  this  subject  were,  however,  published  in 
the  Archives  generates  de  Medecine  and  in  the  Proceedings  of  the  Royal 
Society  of  London  in  1839  and  1841  respectively;  his  later  and  more 
extensive  investigations  appeared  in  the  Proceedings  of  the  California 
Academy  of  Sciences  and  in  the  Comptes  Rendus  of  the  French  Acad- 
emy of  Sciences.  His  inquiries  into  the  effect  of  a  variety  of  inorganic 
salts  when  injected  into  the  circulation  led  to  the  discovery  of  a  number 
of  important  facts;  among  others  to  the  discovery  of  the  Anesthetic 
Action  of  Magnesium  Salts  to  which  much  importance  has  been  ascribed 
in  recent  years.  The  older  generation  of  physiologists,  antedating  the 
modern  development  of  physical  chemistry,  expressed  dosages  and 
concentrations  in  terms  of  the  absolute  weight  of  substance  employed 
and  if,  of  two  substances,  a  smaller  weight  of  one  was  lethal  than  of 
the  other  that  substance  was  deemed  the  more  toxic  of  the  two.  It 
was  Blake  who  first  pointed  out  in  physiological  literature  that  equal 
weights  of  the  various  inorganic  salts  do  not  by  any  means  contain 
equal  numbers  of  molecules,  and  he  urged  that  the  toxicity  or  other 
physiological  actions  of  dissolved  substances  be  referred  not  to  the 
absolute  weight  but  to  the  number  of  gram-molecules  of  .material 
administered.  Proceeding  upon  this  principle  Blake  was  able  to  show 
that  many  substances  hitherto  considered  to  be  of  very  diverse  toxicity 
were  in  reality  very  similar  in  their  physiological  action.  In  particular 
this  was  found  to  be  the  case  with  many  series  of  Isomorphous  Salts 
of  the  metals.  On  the  other  hand  certain  metallic  salts,  hitherto  sup- 
posed to  be  of  like  action  and  toxicity,  were  found,  when  tested  by  this 
new  criterion,  to  differ  very  decidedly  in  their  relative  effect  upon 
living  protoplasm. 


DISTURBANCE  OF  THE  INORGANIC  ENVIRONMENT       311 

The  investigations  thus  initiated  have  been  continued  by  a  very  large 
number  of  subsequent  observers  whose  labors  have  resulted  in  the 
accumulation  of  our  present  extensive  knowledge  of  the  influence 
of  inorganic  salts  upon  living  matter.  A  large  part  of  this  information 
belongs  more  appropriately  to  the  subject  of  Pharmacology  and  we  will 
only  review  it  here  in  so  far  as  it  throws  an  important  light  upon  the 
nature  of  the  chemical  and  physicochemical  factors  which  govern 
the  relationship  of  the  cell  to  its  normal  environment.  The  detailed 
actions  of  the  inorganic  substances  which  are  rarely  if  ever  constituents 
of  the  normal  inorganic  environment  of  protoplasm  we  will  therefore 
not  discuss  beyond  the  enunciation  of  the  brief  generalisation  that  the 
salts  of  the  Heavy  Metals  such  as  silver,  copper,  lead  and  mercury, 
act  as  corrosive  poisons,  leading  to  disintegration  of  the  cells  by  coagu- 
lation and  flocculation  of  the  protoplasmic  proteins,  which  disrupts 
the  continuity  of  the  gel-structure  of  the  cell  and  causes  it  to  break 
up  into  particles  which  fall  apart,  so  that  the  substance  of  the  cell  is 
gradually  eroded  away.  The  compounds  of  Phosphorus,  other  than 
the  phosphates,  and  the  compounds  of  Arsenic  exert  peculiar  effects 
on  metabolism  which  are  fully  described  in  current  works  on  pharma- 
cology. 

The  discovery  that  the  inorganic  salts  which  form  the  normal  con- 
stituents of  the  environment  of  cells  may  under  certain  circumstances 
act  as  protoplasmic  poisons,  is  attributable  in  the  first  place  to  Ringer 
who,  however,  did  not  himself  interpret  his  results  in  this  manner.  He 
found  that  if  the  excised  hearts  or  skeletal  muscles  of  cold-blooded 
animals  be  immersed  in  pure  Sodium  Chloride  solution  which  is  isotonic 
with  blood  serum,  they  lose  their  irritability  and  the  power  of  con- 
traction much  more  rapidly  than  they  do  in  blood  serum,  or  in  solutions 
containing  sodium,  potassium  and  calcium  chlorides  in  the  proportions 
in  which  they  are  present  in  blood-serum.  This  was  at  first  interpreted 
to  mean  that  potassium  and  calcium  were  required  by  these  tissues 
for  nutritive  purposes,  but  later  investigations  have  clearly  shown  that 
while  pure  sodium  chloride  is  definitely  toxic  for  living  tissues,  its 
toxic  properties  are  antagonized  or  annulled  by  a  correct  admixture 
of  potassium  and  calcium  salts. 

All  Inorganic  Salts  in  pure  solutions  exert  in  greater  or  less  concen- 
trations a  toxic  action  upon  protoplasm  which  is  evidenced  by  a  more 
or  less  pronounced  abnormality  of  function.  In  nervous  or  muscular 
tissues  these  effects  are  usually  evidenced  by  an  initial  increase  in 
irritability  followed  by  a  more  or  less  rapid  decrease  and  final  loss  of 
irritability.  Thus,  if  the  foot  of  a  decapitated  frog  be  dipped  into 
solutions  of  various  salts  the  increase  of  irritability  of  the  sensory  nerve 
endings  leads  to  a  reflex  withdrawal  of  the  foot  from  the  solution.  The 
following  were  the  concentrations  of  various  inorganic  substances 
which  Loeb  found  to  be  effective  in  giving  rise  to  this  reflex: 


312         PROPERTIES  OF  THE  COLLOIDAL  CONSTITUENTS 

Mineral  effective 
Substance,  concentration. 

HC1 m/240 

NaOH w/so 

AgNOs •        .  ••-    v.   •••-'  m/iso 

FeCla ...',....      ....      ;••  •       "Veo 


HgCl2 
Aid. 

CaCl2 

SrCl2 

BaCh 

MgCl2 

KC1 

NH4C1 

NaCl 

LiCl 

Sodium  oxalate 

Sodium  citrate 


to 


Vi 


The  effects  observed  were  not  attributable  to  the  imbibition  of  water 
from  these  hypotonic  solutions  because,  it  will  be  recollected,  the  skin 
of  the  frog  is  not  permeable  to  water  in  the  direction  exterior  ->  tissues, 
but  only  in  the  opposite  direction.  The  only  hypertonic  solutions 
employed,  in  which  osmotic  phenomena  might  have  played  a  role, 
were  those  of  the  chlorides  of  the  alkalies  and  ammonium. 

It  was  first  discovered  by  Biedermann  in  1880  that  solutions  of 
certain  Sodium  Salts  cause  skeletal  muscles  which  may  be  immersed 
in  them  to  enter  into  more  or  less  rhythmic  contractions,  reminiscent 
of  those  of  heart-muscles  in  normal  serum  or  in  Ringer's  solution.  He 
also  pointed  out  that  since  these  contractions  continue  to  take  place  in 
the  presence  of  Curare,  which  paralyzes  the  neuromuscular  junctions, 
the  stimulus  which  evokes  them  must  originate  in  the  muscular  tissues 
themselves,  i.  e.,  the  contractions  are  Myogenic.  This  phenomenon 
was  reinvestigated  by  Loeb,  who  found  the  following  minimal  con- 
centrations of  the  various  sodium  salts  just  sufficed  to  evoke  the  semi- 
rhythmic  contractions  in  frogs'  muscles,  the  solutions  being  rendered 
isotonic  with  amphibian  serum  by  the  addition  of  sugar  or  urea. 

Minimal  effective 

Substance.  concentration. 

Sodium  chloride     .      ...      .      «...      . m/ie 

Sodium  bromide    .      .      .      .      ...      .      ;'"'..     .•      .      .      .      .  m/u  to  m/s2 

Sodium  iodide        .      .      .      .      .'    .      .      .     '.      ,      .      r     .      .  m/32 

Sodium  carbonate       .      .      .      »      .      .  • m/ie  to  m/32 

Sodium  sulphate    .      .      .      .      . m/32 

Sodium  acetate      .      .      .     -. w/32  to  m/64 

Sodium  fluoride TO/64  to  OT/36 

Sodium  formate m/80  to  w/ieo 

Sodium  oxalate TO/26o  to  m/3oo 

Sodium  phosphate .  TO/i28  to  m/266 

Sodium  citrate "»/2oo 

It  will  be  observed  that  the  most  efficient  stimulators  in  this  series 
are  the  salts  of  sodium  combined  with  an  acid  (oxalic,  phosphoric, 
citric,  hydrofluoric)  which  precipitates  Calcium  from  its  solutions. 


DISTURBANCE  OF  THE  INORGANIC  ENVIRONMENT       313 

It  is  a  very  significant  fact,  therefore,  that  the  addition  of  traces  of 
soluble  calcium  salts  to  any  of  these  solutions,  whether  containing 
a  calcium-precipitating  acid  or  not,  results  in  prompt  suppression  of 
the  contractions.  Evidently  the  calcium  is  not  required  in  these  cases 
to  supply  a  nutrient  to  the  muscular  tissues,  but  to  antagonize  an 
action  of  an  excess  of  Sodium  which  results  in  abnormality  of  function. 
The  simultaneous  action  of  an  excess  of  sodium  ions  and  a  calcium- 
precipitating  anion  is  more  effective  than  excess  of  sodium  alone, 
because  the  calcium  already  present  in  the  tissues  partially  antagonizes 
the  excess  of  sodium  in  the  environment.  These  facts  led  Loeb  to 
emphasise  the  importance  of  the  ^|  ratio  in  living  tissues  and  in 
their  environment.  Any  pronounced  increase  in  this  ratio  leads  to 
hyperirritability  of  nervous  and  muscular  tissues  and,  in  fact,  as  Loeb 
has  pointed  out,  it  is  only  the  calcium  in  our  blood  and  tissue-fluids 
which  prevents  all  our  skeletal  muscles  from  beating  rhythmically 
like  the  heart. 

The  fact  that  the  heart  continues  to  beat  rhythmically  in  the  presence 
of  the  calcium  in  the  blood,  although  the  skeletal  muscles  cannot  do  so, 
draws  our  attention  to  the  very  important  fact  that  the  effect  of  the 
inorganic  environment  differs  in  different  types  of  living  tissues.  This 
fact  is  very  strikingly  illustrated  by  the  effects  of  salt  solutions  upon 
different  parts  of  the  swimming-bells  of  jellyfish.  These  bells,  in 
normal  sea-water,  are  almost  constantly  contracting  in  a  rhythmic 
manner  and  it  is  by  the  rhythmic  impetus  created  by  the  expelled  water 
that  the  jellyfish  propels  itself  through  the  water.  It  was  first  pointed 
out  by  the  English  Biologist  Romanes  that  when  the  swimming-bell 
of  the  jellyfish  Gonionemus  is  deprived  by  section  of  its  margin,  the 
center  of  the  bell  will  no  longer  beat  in  sea-water,  while  the  margin 
continues  as  before  to  beat  rhythmically.  Since  the  margin  contains 
all  the  nervous  ganglia  of  the  bell  Romanes  concluded  that  the  beats 
of  the  swimming-bell  were  initiated  and  regulated  by  these  nervous 
tissues.  Loeb,  however,  found  that  if  the  centers,  with  the  margin 
excised,  be  placed  in  pure  sodium  chloride  or  sodium  bromide  solutions 
which  are  isotonic  with  sea-water,  they  will  beat  rhythmically  just 
as  the  intact  bell  does  in  sea-water.  The  experiment  really  indicates, 
therefore,  that  the  optimal  salt-mixture  for  rhythmic  excitation  differs 
in  the  nervous  and  the  contractile 'tissues  of  the  bell. 

Another  experiment  which  illustrates  in  a  very  striking  manner 
the  differing  effects  of  the  inorganic  environment  upon  tissues  of 
diverse  function  is  the  following:  When  the  last  abdominal  segment 
of  a  recently-killed  fly1  is  torn  out  with  a  pair  of  forceps  a  length 
of  intestine  is  usually  extracted  from  the  abdomen.  The  muscular 
tissue  in  the  intestines  of  the  Insecta,  unlike  ours,  is  striated.  If  this 
be  wetted  with  M/6  sodium  chloride  solution  and  examined  under  a 

1  The  species  actually  employed  was  the  large  Australian  "bluebottle,"  Callophora 
rillosa. 


314        PROPERTIES  OF  THE  COLLOIDAL  CONSTITUENTS 

microscope,  rhythmic  contractions  will  be  seen  travelling  in  rapid 
succession  from  the  upper  to  the  lower  end  of  the  intestine.  On 
touching  the  intestine  at  about  the  middle  point  with  a  finely  pointed 
camePs-hair  brush  wetted  with  M/6  calcium  chloride  solution  the 
rhythmic  contractions  in  the  affected  area  are  immediately  suppressed, 
but  on  following  a  wave  of  contraction  with  the  eye  as  it  enters  this- 
area  and  disappears,  it  can  be  seen  to  re-issue  below  the  affected  area 
at  the  moment  when  it  would  have  appeared,  had  the  contraction 
actually  traversed  that  section  of  the  intestine.  Evidently,  while  the 
contractile  function  has  been  suppressed,  the  conductive  functions  of 
the  tissues  are  still  unimpaired.  If,  however,  the  middle  part  of 
the  intestine  is  touched  with  potassium  chloride  solution  instead  of 
calcium  chloride  solution,  both  conduction  and  contraction  are  sup- 
pressed and  rhythmic  contractions  remain  confined  to  the  region  above 
the  affected  area,  the  region  below  remaining  quiescent.  Conduction 
and  contraction  are  therefore  very  diversely  affected  by  these  inorganic 
salts. 

EFFECTS  OF  REMOVAL  OF   CALCIUM  FROM  THE   TISSUES  AND 

TISSUE-FLUID. 

We  have  seen  that  an  increase  in  the  Q-*  ratio  in  excised  muscular 
tissues  leads  to  hyperirritability  of  the  tissues  and  that  the  ratio  may 
be  increased  in  either  of  two  ways,  namely  by  increasing  the  concen- 
tration of  the  Sodium  Ions  in  the  environment  or  by  decreasing  the 
Calcium  Ions  by  employing  a  salt  of  which  the  acid  component  either 
precipitates  calcium  or  forms  a  sparingly  dissociated  compound  with  it. 

The  effects  of  injection  of  calcium  precipitants  such  as  citrates, 
oxalates,  fluorides,  tartrates,  oleates  and  other  soaps,  etc.,  are  very 
widespread  and  fundamental.  They  are  traceable  to  muscular,  ner- 
vous and  glandular  tissues.  In  small  doses  whether  taken  by  mouth 
or  injected  intravenously,  they  act  as  Cathartics,  inducing  enhanced 
peristalsis  and  the  evacuation  of  fluid  f eces.  In  larger  do  ses  we  obtain, 
in  addition  to  purgation,  peripheral  twitchings,  i.  e.,  irregular  involun- 
tary contractions  of  the  muscles  of  the  extremities.  An  effect  upon 
the  renal  epithelium  is  also  evidenced  by  a  marked  Diuresis,  or  abnormal 
volume  and  dilution  of  the  urine.  In  still  larger  doses  a  very  curious 
complex  of  symptoms  is  elicited.  Shortly  after  the  injection  of  massive 
doses  of  sodium  citrate  subcutaneously  in  rabbits,  peripheral  twitch- 
ings  occur  which  are  rapidly  succeeded  by  convulsive  movements  and 
marked  disturbances  of  equilibrium.  The  forelegs  are  stiffly  extended 
and  continually  shuffling  forward  with  a  motion  resembling  an  effort 
to  maintain  equilibrium  upon  a  slippery  or  moving  surface.  The  head 
is  thrown  back  and  the  jaws  are  continuously  chewing.  Not  infre- 
quently the  animals  throw  themselves  into  backward  somersaults. 
Exactly  the  same  effects,  without  the  peripheral  twitchings,  purgation 
or  diuresis,  are  obtained  if,  instead  of  administering  massive  doses  to 


REMOVAL  OF  CALCIUM  FROM  THE  TISSUES  315 

the  whole  animal,  minute  doses  are  applied  to  the  White  matter  of  the 
Cerebellum,  by  direct  injection  below  the  gray  matter  of  the  surface. 
The  convulsive  effects  of  large  doses  are  therefore  attributable  to 
excitation  of  the  white  matter  of  the  cerebellum.  Chronic  ingestion 
or  injection  of  calcium  precipitants  leads  to  partial  solution  of  the  bones 
which  become  thin  and  soft,  a  condition  not  infrequently  met  with  in 
sheep  that  have  been  feeding  for  some  time  upon  plants  of  the  Oxalis 
group.  The  effects  of  calcium  precipitants  in  the  order  in  which  they 
occur  are  therefore:  1.  Purgation.  2.  Peripheral  muscular  twitchings 
and  diuresis,  3.  Cerebellar  excitation.  4.  Partial  solution  of  the  bones. 
A  measure  of  Tolerance  to  the  first  three  groups  of  effects  is  developed 
after  repeated  administration. 

The  variety  of  sensitiveness  and  response  of  differing  tissues  to 
modifications  of  the  inorganic  environment  is  again  displayed  in  these 
effects.  The  very  striking  instance  of  the  varying  sensibility  of  differ- 
ent cells  to  this  type  of  environmental  disturbance  is  afforded  by  the 
complete  insensitiveness  of  the  Gray  Matter  of  the  central  nervous 
system  to  calcium  precipitants.  We  have  noted  above  that  the  cere- 
bellar  effects  of  calcium  precipitants  are  only  elicited  when  the  salt 
reaches  the  white  matter  of  the  cerebellum,  and  Maxwell  has  shown 
that  solutions  of  the  various  calcium  precipitants  are  without  effect 
when  placed  upon  the  surface  of  the  motor-area  of  the  cerebrum,  but 
immediately  induce  convulsions  when  they  penetrate  by  diffusion  or 
injection  to  the  underlying  white  fibers. 

The  origin  of  the  purgation  by  the  Saline  Cathartics  has  been  the 
subject  of  much  and  prolonged  discussion.  The  earliest  suggestion 
was  that  made  by  Poiseuille  and  Liebig,  to  the  effect  that  the  action 
of  these  cathartics  was  a  purely  osmotic  one;  the  excess  of  salt  within 
the  intestinal  cavity  withdrawing  water  from  the  tissues  and  tissue- 
fluids,  while  the  tension  of  the  intestinal  musculature  caused  by  this 
collection  of  fluid  within  the  lumen  of  the  intestine  led  to  rapid  expul- 
sion of  the  contents.  The  great  French  Physiologist,  Claude  Bernard, 
however,  showed  that  the  intravenous  injection  of  sulphates  caused 
purgation,  although  the  osmotic  effect  in  this  instance  should  be  the 
reverse  of  that  imagined  by  Poiseuille  and  Liebig,  and  water  should 
be  withdrawn  from  the  intestine  into  the  circulation.  To  meet  this 
objection  a  modification  of  the  osmotic  theory  was  subsequently 
brought  forward  by  Wagner  and  Schmiedeberg,  who  suggested  that 
the  saline  cathartics  modify  the  Permeability  of  the  intestinal  epithe- 
lium, in  such  a  manner  that  the  absorption  of  water  from  the  intestine 
is  hindered  and  the  fluidity  of  the  contents  and  distention  of  the  mus- 
cular walls,  which  ensues  from  the  accumulation  of  unabsorbed  fluids, 
leads  to  the  rapid  evacuation  of  fluid  feces. 

The  discovery  by  Loeb  that  those  salts  which  increase  the  -^  ratio, 
and  especially  those  which  precipitate  calcium,  induce  hyperirri- 
tability  in  muscular  tissues,  at  once  threw  a  new  light  upon  the  action 
of  the  saline  cathartics.  A  large  proportion  of  these  cathartics  are 


316         PROPERTIES  OF  THE  COLLOIDAL  CONSTITUENTS 

sodium  or  magnesium  salts  of  acids  such  as  sulphuric,  carbonic,  phos- 
phoric, citric  or  tartaric  acids  which  form  insoluble  or  sparingly  dis- 
sociated compounds  with  calcium,  and  their  action  in  stimulating  the 
muscles  of  the  intestine  may  be  regarded  simply  as  an  instance  of  a 
general  effect  upon  contractile  tissues.  Barium  Chloride,  which  stands 
in  the  peculiar  position  of  not  being  a  calcium  precipitant,  and  never- 
theless being  a  powerful  stimulant  of  muscular  tissues,  is  also  a  very 
drastic  purgative.  The  inorganic  reagents  which  induce  contractions 
in  excised  skeletal  muscles,  therefore,  cause  purgation  when  adminis- 
tered by  mouth  or  injected  intravenously. 

Continuing  and  extending  the  above-cited  investigations  of  Claude 
Bernard,  J.  B.  Macallum  showed  that  if  10  c.c.  doses  of  M/6  sodium 
citrate,  sulphate  or  tartrate  be  administered  subcutaneously  to  rabbits, 
followed  ten  minutes  later  by  5  c.c.,  and  ten  minutes  after  that  by  5  c.c. 
more,  very  pronounced  purgation  follows.  Purgation  may  also  be  induced 
by  perfusion  of  these  solutions  into  the  bloodvessels  supplying  a  loop  of 
intestine,  or  even  by  painting  the  solution  upon  the  peritoneal  surface  of 
the  intestine.  By  whatever  avenue  the  salt  reaches  the  muscular  tissue, 
therefore,  contractions  are  induced.  This  disposes  of  the  original  osmotic 
theory.  The  theory  of  Wagner  and  Schmiedeberg,  that  the  fluidity  of 
the  feces  induced  by  these  salts  is  due  to  the  non-absorption  of  water 
from  the  intestine,  was  shown  to  be  unnecessary  by  the  discovery  by 
Macallum  that  part,  at  least,  of  the  fluidity  of  the  feces  is  attributable 
to  the  active  secretion  of  fluid  from  the  mucous  glands  of  the  intestine 
into  its  lumen.  Thus,  a  loop  of  intestine  about  30  centimeters  long  in 
a  rabbit  was  thoroughly  cleaned  out  by  compression  and  the  ends  tied. 
From  time  to  time  before  and  after  the  administration  of  a  cathartic 
salt  the  loop  was  opened  and  the  content  of  fluid  determined.  The 
following  is  an  illustrative  result: 

Loop  contained  at  the  beginning    .      .      .      .      .      .      .      .      .      .  5.0  c.c. 

Fluid  secreted  in  the  first  ten  minutes 0.2    " 

Fluid  secreted  in  the  second  ten  minutes 0.5    " 

2  c.c.  of  m/»  barium  chloride  injected  subcutaneously. 

Fluid  secreted  in  the  first  ten  minutes  after  injection     .      .      .      .  4.0  c.c. 

Fluid  secreted  in  the  second  ten  minutes  after  injection      .      .      .  3.4    " 

Fluid  secreted  in  the  third  ten  minutes  after  injection  .      .      .      .  3.0" 

the  loop  after  the  administration  of  the  barium  chloride,  also  under- 
went powerful  contractions. 

Even  when  the  saline  cathartic  is  administered  by  mouth,  the 
operative  portion  of  it  is  that  which  reaches  the  intestine  through  the 
medium  of  the  circulation,  so  that  even  in  this  instance  an  osmotic 
effect  of  the  salt  is  excluded.  This  has  been  very  strikingly  shown  by 
the  experiments  of  Hertz,  Cook  and  Schlesinger,  conducted  in  Guy's 
Hospital  in  London.  These  observers  employed  human  subjects  for 
their  experiments,  following  the  passage  of  the  cathartic  down  the 
intestine  by  simultaneously  administering  bismuth  oxychloride  and 
following  the  shadow  cast  by  this  substance  on  an  .r-ray  plate.  Sepa- 


REMOVAL  OF  CALCIUM  FROM   THE  TISSUES  317 

rate  experiments  upon  a  patient  with  an  iliac  fistula  showed  that  the 
cathartic  and  the  bismuth  oxychloride  travelled  down  the  intestine 
together,  i.  e.,  the  cathartic  did  not  reach  any  point  of  the  intestine  in 
appreciable  advance  of  the  shadow  cast  upon  the  plate. 

Three  persons  received  two  ounces  each  of  bismuth  oxychloride  i  n 
half  a  pint  of  cold  water  at  8  A.M.  Breakfast  was  given  at  8.30.  Cecal 
sounds  were  heard  and  a  shadow  appeared  in  the  cecum  four  hours 
after  the  meal. 

Two  days  later  the  same  persons  received  a  Seidlitz  powder  with 
the  same  mixture.  The  shadow  appeared  in  the  cecum  at  the  usual 
time,  namely,  four  hours  after  the  meal,  but  while  normal  feces  were 
passed  before  breakfast,  fluid  stools,  due  to  the  cathartic,  were  passed 
at  9.15,  9.40  and  9.45  respectively,  no  less  than  three  hours  before  the 
first  trace  of  bismuth  or  of  the  saline  cathartic  reached  the  cecum  by 
the  way  of  direct  passage  through  the  intestine. 

The  same  conclusion  was  reached  by  chemical  analysis  of  the  feces, 
a  sulphate  being  in  this  instance  employed  as  the  cathartic  salt: 

Per  cent.  Per  cent. 

Feces.                                                       of  water.  Total  SO4  of  SO4. 

First  day  normal 80.9                     0.037  0.045 

Second  day  normal  (10.15)      .      .      .      80.0                     0.016  0.032 

Second  day  watery  (11.25)       .      .      .      91.1                      0.091  0.041 

Third  day  normal 77.3                     0.270  0.220 

Thus  the  watery  feces  evacuated  in  response  to  the  cathartic  con- 
tained very  little  more  sulphate  than  the  normal  feces  of  the  pre- 
ceding day,  while  the  normal  feces  of  the  day  following  the  purgation 
contained  less  than  the  normal  percentage  of  water,  and  a  great  excess 
of  sulphates.  Were  either  Liebig's  or  Wagner  and  Schmiedeberg's 
hypothesis  the  correct  interpretation  of  the  facts,  we  would  expect 
these  feces  to  be  very  fluid,  whereas  the  experiment  shows  that  the 
sulphate  that  remains  unabsorbed  is  actually  much  less  efficient  in 
promoting  peristalsis  than  the  proportion  which  circulates  in  the  blood- 
stream. That  an  excess  of  sulphates  were  actually  circulating  in  the 
blood-stream  while  purgation  was  taking  place  is  evidenced  by  the 
fact  that  the  urine  collected  between  8  A.M.  and  4  P.M.  on  the  second 
day  contained  0.624  grammes  more  S04  than  the  urine  collected  during 
the  same  period  on  the  previous  day. 

It  must  be  admitted  that  the  purgative  action  of  the  saline  cathar- 
tics is  not  to  be  entirely  accounted  for  by  the  precipitation  of  the 
calcium  in  the  tissues,  since  Barium  and  Magnesium,  irrespective  of 
whether  they  are  combined  with  calcium  precipitating  acids  or  not, 
will  induce  purgation.  The  possibility  must  be  borne  in  mind,  how- 
ever, although  we  as  yet  possess  no  direct  evidence  which  bears  upon 
it,  that  barium  and  magnesium,  being  related  divalent  metals,  may 
possibly  displace  calcium  from  certain  compounds  in  the  protoplasm 
of  the  tisues  affected,  and  in  this  connection  it  is  perhaps  significant 
that  the  urinary  output  of  calcium  runs  parallel  to  the  output  of 


318         PROPERTIES  OF  THE  COLLOIDAL  CONSTITUENTS 

magnesium.  Furthermore  both  the  cathartic  and  the  anesthetic 
actions  of  magnesium  salts  are  antagonized  and  annulled  by  the 
administration  of  calcium  salts.  At  all  events  barium  salts  share  with 
the  calcium  precipitants  the  common  property  of  inducing  hyperirri- 
tability  in  muscular  tissues,  while  the  exceptional  sensitiveness  of  the 
intestinal  musculature  to  magnesium  may  be  perhaps  regarded  as 
affording  another  instance  of  the  diverse  susceptibility  of  the  various 
types  of  tissue-cells  to  the  influence  of  changes  in  the  inorganic  environ- 
ment. That  the  intestinal  musculature  is  not  the  only  tissue  which 
is  profoundly  affected  by  magnesium  salts  is  shown  by  the  fact  that 
the  introduction  of  a  considerable  excess  of  magnesium  chloride  into 
the  blood-stream  induces  Glycosuria  in  rabbits. 


THE  MUTUALLY  ANTAGONISTIC  ACTION  OF  SALTS,  AND  PHYSIO- 
LOGICALLY BALANCED  SOLUTIONS. 

We  have  already  seen  that  a  small  proportion  of  calcium  inhibits 
the  action  of  sodium  salts  in  inducing  rhythmic  pulsations  of  many 
contractile  tissues.  This  is,  however,  merely  a  particular  instance  of  a 
very  general  phenomenon,  as  the  investigations  of  Loeb  in  animal 
physiology  and  of  Osterhout  in  plant-physiology  have  most  abundantly 
demonstrated. 

For  example,  the  fertilized  eggs  of  the  marine  fish  Fundulus  will 
develop  normally  in  distilled  water.  Inorganic  salts  are  therefore  not 
necessary  for  their  nutrition.  They  will  also  develop  normally,  of 
course,  in  sea-water  in  which  the  various  saline  constituents  other  than 
bicarbonates  and  phosphates  are  present  in  approximately  the  following 
concentration  and  proportion  (Van  t'Hoff's  solution) : 

5/8m  sodium  chloride 1000  parts  by  volume 

5/8m  magnesium  chloride 78      "  " 

5/8m  magnesium  sulphate 38      "  " 

5/8m  potassium  chloride • .      .      .      .          22      "  " 

5/8m  calcium  chloride  t 10      "  " 

In  |  m  sodium  chloride  solution,  without  the  admixture  of  the  other 
salts,  however,  the  eggs  will  not  live  for  more  than  twelve  hours, 
despite  the  fact  that  this  solution  is  isotonic  with  sea-water.  Evidently 
sodium  chloride  is  definitely  toxic  for  these  organisms.  In  the  following 
mixtures: 

96  c.c.  of  5/8m  NaCl  +  4  c.c.  of  5/8m  MgCl2 
96  c.c.  of  5/8m  NaCl  +  4  c.c.  of  S/Sm.KCl 
96  c.c.  of  5/8m  NaCl  +  4  c.c.  of  5/8m  CaCl2 

they  will  live  only  for  about  twenty-four  hours  or  even  less.    When  the 
eggs  are  placed  in  the  following  solutions: 

96  c.  c.  of  5/8m  NaCl  +  2  c.c.  of  5/8m  MgCh  +  2  c.c.  of  5/8m  CaCl2 
96  c.c.  of  5/8m  NaCl  +  2  c.  c.  of  5/8m  MgCl2  +  2  c.c.  of  5/8m  KC1 
96  c.c.  of  5/8m  NaCl  +  2  c.c.  of  5/8m  CaCl2    +  2  c.c.  of  5/8m  KC1 


MUTUALLY  ANTAGONISTIC  ACTION  OF  SALTS  319 

the  eggs  live  for  less  than  thirty  days  in  the  first  two  solutions,  but  in 
the  third,  which  corresponds  in  composition  with  a  concentrated 
Ringer's  Solution,  the  eggs  live  for  an  indefinite  period  and  develop 
normally.  Evidently  the  toxic  properties  of  sodium  chloride  are 
neutralized  or  antagonized  by  admixture  with  a  small  proportion  of 
other  inorganic  salts.  When  the  correct  mixture  is  obtained  the  solu- 
tion is  devoid  of  toxicity  and  we  speak  of  it  as  a  "Physiologically 
Balanced"  salt  solution.  Evidently  Ringer's  solution  and  sea-water  are 
physiologically  balanced  solutions  in  so  far  as  the  tissues  of  Fundulus 
are  concerned. 

Sodium  chloride  is  not  peculiar  in  exerting  a  toxic  effect  in  pure 
solution.  In  fact  it  may  be  said  that  any  salt  without  admixture 
with  other  salts  is  more  or  less  toxic  for  living  protoplasm.  This  is 
very  clearly  demonstrated  by  the  following  among  very  many  experi- 
ments of  this  character  which  we  owe  to  Loeb. 

Antagonism  between  sodium  chloride  and  zinc  sulphate  (Fundulus). 


Percentage  of 
-  eggs  which 

Solution.  develop: 

lOOc.c.  H2O       .................     49 

lOOc.c.  H2O      +     8  c.c.  l/32m  ZnSO4    ..........        0 

100  c.c.  8/8m  NaCl  +  8  c.c.  l/32m  ZnSO4   ........      .        1 

100  c.c.  7/8m  NaCl  +  8  c.c.  l/32m  ZnSO4   .........        6 

100  c.c.  6/8m  NaCl  +  8  c.c.  l/32m  ZnSO4  .........        8 

100  c.c.  5/8m  NaCl  +  8  c.c.  l/32m  ZnSO4   .........      29 

100  c.c.  4/8m  NaCl  +  8  c.c.  l/32m  ZnSO4   .........      34 

100  c.c.  3/8m  NaCl  +  8  c.c.  l/32m  ZnSO4  .........      37 

lOOc.c.  2/8m  NaCl  +  8  c.c.  l/32m  ZnSO4  .........      38 

Evidently  a  very  dilute  solution  of  zinc  sulphate  is  highly  toxic  for 
Fundulus  eggs.  Sodium  chloride  in  excess  is  also  very  toxic.  A  definite 
admixture  of  these  two  toxic  salts  may  be  found,  however,  which  is 
almost  devoid  of  toxicity. 

In  the  above  experiment  we  have  an  instance  of  antagonism  between 
a  monovalent  metal  and  a  divalent  metal.  Antagonism  may  also 
be  displayed  between  two  monovalent  metals  or  between  two  divalent 
metals.  The  following  is  an  illustrative  example: 

Antagonism  between  magnesium  chloride  and  strontium  chloride 
(Fundulus)  . 

Percentage  of 

eggs  which 

Solution.  develop: 

100  c.c.  5/8m  MgCl2     .............  0 

100  c.c.  5/8m  MgCl2  +  1  c.c.  of  5/8m  SrCl2       .....  '    .  25 

lOOc.c.  5/8m  MgCl2  +  2  c.c.  of  5/8m  SrCl2        ......  22 

100  c.c.  5/8m  MgCl2  +  3  c.c.  of  5/8m  SrCl2        ......  9 

lOOc.c.   5/8m  MgCl2  +  4  c.c.  of  5/8m  SrCl2        ......  0 

lOOc.c.  5/8m  MgCl2  +  5  c.c.  of  5/8m  SrCl2        ........        0 

Similar  antagonism  was  found  to  subsist  between  lithium  chloride 
and  zinc  sulphate,  potassium  chloride  and  zinc  sulphate,  ammonium 
chloride  and  zinc  sulphate,  sodium  acetate  and  lead  acetate,  sodium 
chloride  and  manganese  chloride,  sodium  chloride  and  cobalt  chloride, 


320         PROPERTIES  OF  THE  COLLOIDAL  CONSTITUENTS 

sodium  chloride  and  lead  acetate,  sodium  chloride  and  aluminium  sul- 
phate, sodium  chloride  and  chromium  sulphate,  potassium  chloride  and 
calcium  nitrate  and,  in  fact,  some  measure  of  mutual  antagonism  is 
usually  but  not  invariably  found  to  subsist  between  every  pair  of  inor- 
ganic salts. 

Very  striking  examples  of  the  mutual  antagonism  of  inorganic 
salts  are  afforded  by  the  experiments  of  Osterhout  upon  plant-tissues. 
The  following  shows  the  aggregate  length  of  roots  produced  after  sixty 
days  by  wheat-seeds  allowed  to  germinate  in  various  salt  solutions  of 
2^  molecular  concentration. 

Aggregate  length 
Solution.  of  roots  mm. 

Sodium  chloride        .      .      .  '  .      .  "  .      .'.     .      .      . 59 

Potassium  chloride         .      .     .,      .      .  ''. 68 

Magnesium  chloride „     .-.:'.. 7 

Calcium  chloride 70 

1000NaCl+  10CaCl2       .      .      .      .      ; 254 

lOOONaCl  +  22KC1  +  10CaCl2 324 

1000  NaCl  +  78MgCl2  +  10CaCl2 377 

1000  NaCl  +  78MgCl2  +  38MgSO4  +  22KC1  +  10CaCl2       .      .      . .    .   360 
Distilled  water 740 

Since  the  roots  exhibit  a  maximum  growth  in  distilled  water  the 
various  salts  are  evidently  not  required  for  the  nutrition  of  the  plants. 
The  individual  salts  in  pure  solution  are  all  highly  toxic  as  compared 
with  distilled  water,  but  mixtures  of  the  salts  in  proportions  approxi- 
mating to  those  found  in  sea-water  permit  very  extensive  growth 
of  roots  to  occur.  The  following  data  show  the  percentage-increase  in 
the  length  of  the  thallus  which  develops  from  the  seeds  of  Equisetum 
in  various  salt  solutions  of  Tf  ^  molecular  concentration. 

Percentage  increase 
in  length  of  thallus 
Solution.  after  50  days. 

Sodium  chloride »      .      .      .        0 

Potassium  chloride 0 

Magnesium  chloride 0 

Calcium  chloride 700 

lOOONaCl  +  22KC1 0 

lOOONaCl  +  78MgCl2 40 

lOOONaCl  +  78MgCi2  +  22KC1 40 

lOOONaCl  +  10CaCl2 980 

lOOONaCl  +  22KCI  +  10CaCl2  . 1500 

lOOONaCl  +  78MgCl2  +  10CaCl2 1760 

lOOONaCl  +  78MgCl2  +  38MgSO4  +  22KC1  +  10CaCl2      ...        1500 
Distilled  water 1760 

In  certain  instances  the  toxicity  of  such  a  universally  distributed 
substance  as  sodium  chloride  may  be  extremely  great.  Thus  Osterhout 
found  specimens  of  Vaucheria  which  were  killed  within  a  few  days  by 
so  small  a  concentration  as  y^^  sodium  chloride,  although  the  running 
water  in  which  these  algse  were  growing  contained  no  less  than  twelve 
times  this  concentration  of  sodium  chloride.  In  the  brook,  however, 
the  toxicity  of  the  sodium  chloride  was  completely  annulled  by  the 


MUTUAL  ANTAGONISM  OF  INORGANIC  SALTS  321 

traces  of  other  salts,  magnesium,  potassium  and  calcium  chlorides 
which  the  water  contained. 

Similar  phenomena  of  antagonism  have  been  observed  by  C.  B. 
Lipman  in  culture-media  containing  bacteria.  In  certain  cases,  how- 
ever, no  mutual  antagonism  was  observed,  as  in  the  case  of  magnesium 
and  calcium  salts  acting  upon  Bacillus  subtilis.  Furthermore,  although 
the  toxicities  of  potassium  chloride  and  calcium  chloride  for  this,  and 
probably  for  other  am  nonifying  bacteria,  are  mutually  diminished  by 
their  admixture,  this  is  not  the  case  for  sodium  and  calcium  chlorides,  a 
mixture  of  these  two  salts  being  more  toxic  for  all  proportions  of  cal- 
cium than  sodium  chloride  alone.  These  exceptional  phenomena  appear 
to  differentiate  the  ammonifying  bacteria  very  sharply  from  other  types 
of  living  tissue. 

The  mutually  antagonistic  toxicity  of  inorganic  salts  is  therefore  a 
phenomenon  which  is  universally  displayed  whatever  type  of  proto- 
plasm we  employ.  In  certain  types,  as  those  afforded  by  the  ammoni- 
fying bacteria,  certain  antagonisms  may  fail  to  be  exhibited,  but 
other  pairs  of  salts,  again,  will  clearly  annul  each  other's  toxicity.  We 
may  infer  therefore,  that  the  toxicity  of  pure  salts  for  protoplasm  is  a 
universal  property,  and  that  in  the  majority  of  instances  a  mixture 
of  any  two  salts  is  less  toxic  than  either  of  the  components  alone.  It 
is  certainly  not  an  accident  that  for  all  the  forms  of  life  which  have 
been  investigated,  the  most  nearly  innocuous  mixtures  correspond  in 
composition  either  to  sea-water  (Van  t'Hoff's  solution)  or  to  Ringer's 
solution.  In  these  mixtures  of  five  and  three  salts  respectively  the 
annulment  of  toxicity  is  far  more  complete  than  in  any  binary  mixture. 
Sea-water  and  Ringer's  solution  are  therefore,  Physiologically  Balanced 
Solutions,  but  for  certain  of  the  higher  animals,  for  example  in  the 
mammals  of  which  the  tissues  are  adapted  to  an  environment  having 
the  composition  of  Ringer's  solution,  sea-water,  as  it  is  composed  today, 
is  no  longer  a  physiologically  balanced  solution.  The  determination 
of  the  physiological  balance  depends,  therefore,  upon  the  properties 
of  the  protoplasm  upon  which  the  salts  are  acting  and  not  upon  any 
peculiar  properties  of  the  salt-mixture  in  question,  such  as  double  salt- 
formation,  etc. 

THE   ORIGIN   OF   THE   MUTUAL   ANTAGONISM   OF    INORGANIC 

SALTS. 

Since  the  mutual  antagonism  of  salts  originates  in  a  property  of 
protoplasm  rather  than  in  any  physical  peculiarity  of  the  salt-mixtures, 
we  are  led  to  infer  that  the  phenomenon  must  probably  be  due  to 
chemical  interactions  between  the  constituents  of  the  salt-mixture  and 
some  constituents  of  the  cells.  Now  antagonism,  as  we  have  seen, 
may  be  displayed  between  almost  any  pair  of  metal  ions,  but  it 
may  also  be  displayed  between  different  pairs  of  acid  radicals.  More- 
over the  toxicity  of  both  acids  and  bases  may  be  partially  annulled  by 
21 


322         PROPERTIES  OF  THE  COLLOIDAL  CONSTITUENTS 

suitable  neutral  salts.  It  is  clear,  therefore,  that  any  constituent  of 
the  cell  which  is  responsible  for  these  phenomena  must  be  capable  of 
entering  into  combination  with  both  acids  and  bases  and  with  both 
the  acid  and  the  basic  radicals  of  salts.  The  Proteins  are  the  only 
abundant  constituents  of  the  cell  which  have  been  demonstrated  to 
possess  these  properties,  and  it  has  therefore  been  inferred  by  Loeb 
and  is  now  very  generally  assumed,  that  the  toxicity  of  sodium  salts, 
for  example,  is  attributable  to  the  formation  of  sodium  proteinates 
which,  if  present  in  too  great  a  proportion  in  the  cell,  confer  upon  the 
protoplasm  properties  which  are  incompatible  with  the  maintenance 
of  normal  function.  The  toxicity  of  calcium  salts  is  regarded  as  being 
attributable  in  like  manner  to  the  undue  predominance  of  calcium 
proteinates  in  the  cell.  An  admixture  of  several  types  of  protein  salts 
is  requisite  to  confer  upon  the  protoplasm  of  the  cell  the  exact  complex 
of  qualities  essential  to  the  maximal  furtherance  of  its  vital  activities. 

Much  light  has  been  thrown  upon  this  question  by  two  very  striking 
series  of  investigations,  namely  the  Flotation  Experiments  of  Loeb  and 
the  Conductivity  Experiments  of  Osterhout.  The  eggs  of  the  marine 
fish  Fundulus  which  were  employed  in  the  earlier  experiments  cited 
above  have  a  specific  gravity  which  is  considerably  in  excess  of  that  of 
sea-water.  They  will  float  in  a  --£-  molecular  solution  of  sodium 
chloride,  while  they  sink  in  a  V~  molecular  solution.  The  experiments 
consisted  in  placing  the  eggs  in  solutions  exceeding  --f-  molecular  in 
concentration,  which  is,  of  course,  considerably  hypertonic  to  the 
contents  of  the  eggs,  and  observing  how  long  they  will  float  in  such 
solutions.  The  withdrawal  of  water  from  the  eggs  is  manifested  not 
only  by  shrinkage  of  volume,  but  by  a  coincident  increase  in  specific 
gravity  which  results  finally  in  the  eggs  acquiring  a  higher  specific 
gravity  than  the  medium  so  that  they  sink  in  it.  Continued  flotation 
in  hypertonic  solutions  is  therefore  evidence  of  impermeability  of  the 
superficies  of  the  cell  for  water. 

It  is  found  that  if  the  eggs  are  placed  in  a  3  molecular  solution  of 
Sodium  Chloride  they  will  float,  but  as  a  rule  not  longer  than  three 
hours.  After  that  they  sink  to  the  bottom  of  the  test-tube  while  the 
loss  of  water  which  has  led  to  their  sinking  is  evidenced  by  collapse  of 
the  egg-membrane,  and  shrinkage  of  the  yolk-sac.  When  the  eggs  are 
placed  in  a  -/  molecular  solution  of  Calcium  Chloride  they  float  at  first, 
but  they  sink  in  about  half  an  hour.  If,  however,  the  eggs  are  placed 
in  a  mixture  of  50  c.c.  of  3  molecular  sodium  chloride  and  2  c.c.  of  --f- 
molecular  calcium  chloride,  they  will  float  for  three  days  or  more  at  the 
surface  of  the  solution,  the  eggs  shrink  but  little  or  not  at  all,  and  the 
embryos  continue  to  live.  In  a  mixture  of  50  c.c.  of  2J  m.  NaCl  +  1 
c.c.  of  2J  m.  KC1  +  0.75  c.c.  of  2J  m.  CaCl2  some  of  the  eggs  will  con- 
tinue to  float  for  as  long  as  ten  days,  while  in  a  2J  m.  solution  of  pure 
sodium  chloride  they  do  not  float  for  more  than  a  few  hours. 

These  phenomena  admit  of  only  one  explanation,  namely,  that  in 
normal  sea-water  the  superficies  of  the  Fundulus  egg  is  practically 


MUTUAL  ANTAGONISM  OF  INORGANIC  SALTS  323 

impermeable  to  water,  but  that  in  a  physiologically  unbalanced  salt 
solution  this  natural  impermeability  is  lost,  and  hence,  if  the  solution 
is  at  the  same  time  hypertonic,  water  diffuses  out  of  the  egg  and  the 
resultant  increase  in  specific  gravity  causes  it  to  sink.  The  same  solu- 
tions which  cause  this  loss  of  water  are  also  toxic  for  the  developing 
embryos.  Hence  the  toxicity  of  unbalanced  solutions  is  associated 
with  an  increased  permeability  of  the  cells. 

The  same  conclusion  has  been  reached  by  Osterhout  in  quite  another 
way.  This  observer  has  employed  the  electrical  conductivity  of  plant- 
tissues  as  a  measure  of  their  permeability  for  ions,  that  is  of  the  resist- 
ance which  the  surfaces  of  the  cells  offer  to  the  transport  of  ions  across 
them.  Discs  about  13  m.m.  in  diameter  were  cut  from  the  fronds  of 
marine'algse  (Laminaria),  the  average  thickness  of  a  frond  being  about 
0.5  m.m.  One  or  two  hundred  of  these  discs  were  then  packed  together 
like  a  roll  of  coins,  into  a  solid  cylinder  of  from  50  m.m.  to  100  m.m. 
in  length.  They  were  held  in  place  by  glass  rods  so  arranged  as  to  make 
a  hollow  cylinder  which  closely  fitted  over  the  outside  of  the  solid  cyl- 
inder of  tissue.  The  spaces  between  the  rods  allowed  free  access  of 
various  salt  solutions  to  the  living  tissue.  At  each  end  of  the  cylinder 
of  tissue  was  placed  a  platinum  electrode  which  could  be  pressed 
firmly  by  means  of  a  screw  against  the  opposite  ends  of  the  cylinder. 
The  conductivity  of  the  cylinder  was  estimated  in  the  usual  way.  The 
surface  in  and  out  of  which  ions  were  forced  by  the  current,  amounted 
to  from  26,000  to  53,000  square  centimeters;  an  increase  in  the  con- 
ductivity of  the  cylinder  implied  decreased  resistance  to  the  passage 
of  ions  across  the  surfaces  of  the  tissue,  i.  e.,  an  increased  permeability 
for  electrolytes,  while  a  decrease  in  the  conductivity  of  the  cylinder 
implied,  on  the  contrary,  decreased  permeability  of  the  cells.  It  will 
be  observed  that  the  permeability  measured  by  Osterhout  was  per- 
meability for  dissolved  electrolytes,  while  that  measured  by  Loeb  was 
permeability  for  water. 

On  transferring  the  cylinder  of  Laminaria  from  sea-water  to  Sodium 
Chloride  solution  of  the  same  temperature  and  conductivity  (0.52 
molecular),  the  resistance  fell  from  the  initial  value  of  1100  ohms  in 
sea-water  to  890  ohms  in  ten  minutes.  In  fifteen  minutes  it  had  fallen 
to  780  ohms,  after  sixty  minutes  to  420  ohms,  and  thereafter  continued 
to  fall  steadily  until  it  reached  a  constant  minimal  value  of  320  ohms, 
which  was  found  to  be  the  resistance  of  a  column  of  sea-water  of  the 
same  length  and  diameter.  In  other  words,  in  pure  sodium  chloride 
solution  the  cell-surfaces  in  Laminaria  increase  in  permeability  until 
finally  they  interpose  no  resistance  at  all  to  the  transference  of  ions 
across  them. 

A  very  striking  contrast  to  this  result  is  obtained  if  a  similar  column 
of  tissue  be  transferred  from  sea-water  to  a  solution  of  Calcium  Chloride 
having  the  same  conductivity  as  sea-water.  In  this  case  the  resistance 
of  the  tissue  initially  rises,  very  often  from  the  initial  sea-water  value 
of  1100  ohms  to  1750  ohms  in  the  first  fifteen  minutes.  The  resistance 


324         PROPERTIES  OF  THE  COLLOIDAL  CONSTITUENTS 

remains  stationary  at  this  level  for  some  hours,  and  then  slowly  sinks 
until  it  finally  reaches  the  level  of  320  ohms,  which  represents  zero 
resistance  on  the  part  of  the  surfaces  within  the  tissue.  The  permea- 
bility of  the  cell-surfaces  in  calcium  chloride  solutions,  therefore,  at 
first  decreases  and  later  increases. 

A  mixture  of  1000  c.c.  of  molecular  sodium  chloride  +  15  c.c.  of 
molecular  calcium  chloride  was  then  diluted  until  it  had  the  same 
conductivity  as  sea-water.  A  similar  column  of  Laminaria  tissue,  when 
placed  in  this  mixture,  neither  gained  nor  lost  resistance,  and  had  the 
same  conductivity  after  twenty-four  hours  as  it  normally  has  in  sea- 
water.  The  antagonistic  action  of  calcium  chloride  upon  the  toxicity 
of  sodium  chloride  is  therefore  seen  to  depend  upon  the  maintenance 
of  the  normal  permeability  of  the  cells. 

In  general  it  was  found  that  while  the  salts  of  monovalent  cations 
such  as  sodium,  potassium,  csesium,  rubidium,  lithium  and  ammo- 
nium increase  the  permeability  of  the  tissue  from  the  beginning,  the 
salts  of  divalent  cations,  such  as  magnesium,  calcium,  barium,  stron- 
tium, manganese,  cobalt,  iron,  nickel,  zinc,  cadmium  and  tin  agree 
in  bringing  about  an  initial  decrease  of  conductivity  followed  by  a 
relatively  gradual  increase.  The  initial  decrease  is,  however,  very 
slight  in  the  case  of  magnesium.  Acids  resemble  the  divalent  cations 
in  causing  an  initial  decrease  followed  by  an  increase  of  permeability, 
but  both  the  increase  and  the  decrease  are  much  more  rapid  than 
they  are  in  solutions  of  neutral  salts  of  divalent  cations.  Alkalies 
resemble  the  salts  of  the  monovalent  cations  in  causing  an  increase  of 
permeability  from  the  first. 

If  the  increase  in  permeability  does  not  exceed  a  certain  limit,  the 
return  of  the  tissue  to  normal  sea-water  results  in  the  restoration  of 
normal  permeability  and  the  tissue  is  not  permanently  injured.  If, 
however,  the  increase  of  permeability  exceeds  this  limit  then  the 
normal  permeability  is  not  recoverable  and  the  attainment  of  absolute 
permeability,  i.  e.,  zero  resistance  of  the  surfaces  of  the  cells  to  the 
passage  of  electrolytes  across  them,  indicates  death  of  the  tissue.  The 
toxic  action  of  pure  salts  is  therefore  seen  to  originate  in  the  irreparable 
impairment  of  the  normal  resistance  which  the  surface  of  the  cell 
opposes  to  the  penetration  or  exit  of  water  and  inorganic  salts. 

Having  regard  to  the  fact  that  the  Proteins  of  the  cell  are  the  only 
abundant  constituents  which  are  capable  of  entering  into  combination 
with  all  of  these  diverse  substances  we  may  assume  that  the  alterations 
of  Permeability  which  attend  immersion  of  living  tissue  in  abnormal 
inorganic  environments  are  due  to  alterations  in  the  physical  consist- 
ency of  the  interstitial  protein  solution  or  jelly  which  holds  the  lipoidal 
elements  in  suspension.  Alterations  in  the  consistency  of  the  Inter- 
stitial Protein  Medium,  and  especially  alteration  of  the  texture  of  the 
spongework  of  which  it  is  composed,  must  necessarily  modify  the 
spacing  of  the  superficial  lipoidal  elements,  and  by  widening  or  narrow- 
ing the  interstitial  pores,  increase  or  decrease  the  permeability  of  the 


MUTUAL  ANTAGONISM  OF  INORGANIC  SALTS  325 

cell  for  water  and  for  substances  which  are  soluble  in  water,  but 
insoluble  in  fats. 

Of  course  the  alteration  of  the  texture  of  the  interstitial  protein 
jelly  which  ensues  when  cells  are  immersed  in  abnormal  inorganic 
media  may  be  expected,  not  only  to  affect  the  permeability  of  the  cells, 
but  also  a  variety  of  other  properties  of  the  cells,  and  in  this  way  to 
affect  a  variety  of  their  functions.  Thus,  as  Loeb  has  pointed  out, 
the  effects  of  diverse  salt  solutions,  and  especially  those  of  calcium 
precipitants  upon  the  phenomena  of  motility,  are  not  solely  and  directly 
to  be  attributed  to  changes  in  the  permeability  of  the  superficies  of 
the  contractile  elements.  Indeed  it  would  be  manifestly  unreasonable 
on  a  priori  grounds  to  make  such  an  assumption.  The  permeability 
of  the  cells  having  been  affected,  however,  the  salts  which  penetrate 
them  induce  further  changes  which  modify  their  performance  of  func- 
tion. This  is  very  clearly  indicated  by  the  following  experiments  in 
which  Loeb  sought  to  ascertain  whether  the  ratio  of  ^  or  of  jjjg~$~C» 
which  is  requisite  for  the  maintenance  of  life  is  the  same  as  that  required 
for  the  maintenance  of  motility.  The  eggs  of  Fundulus  were  immersed 
in  solutions  of  sodium  chloride  of  varying  concentration,  and  the  con- 
centration of  calcium  chloride  which  had  to  be  added  to  each  sodium 
chloride  solution  to  permit  fifty  per  cent,  of  embryos  to  form  was 
determined.  It  was  found  that  if  the  concentration  of  sodium  chloride 
varies  in  the  ratio  1:2:3  the  requisite  additions  of  calcium  chloride 
vary  in  the  proportion  0.3  : 1.3  :  3.2.  In  other  words,  if  we  double  the 
concentration  of  sodium  chloride  we  must  quadruple  the  amount  of 
calcium  chloride,  and  if  we  triple  the  concentration  of  sodium  chloride 
we  must  add  about  ten  times  as  much  calcium  chloride.  To  permit 
normal  development  and  therefore,  presumably,  to  maintain  normal 
Permeability  calcium  chloride  must  be  added  almost  in  the  ratio  of  the 
square  of  the  concentration  of  the  sodium  chloride. 

Now  when  we  turn  to  the  proportion  of  calcium  necessary  for  the 
maintenance  of  unimpaired  Motility  we  find  a  very  different  relation- 
ship obtaining.  For  this  investigation  the  newly  hatched  larva?  of  a 
barnacle  (Balanus  eburneus)  were  employed.  These  larvae  are  incessant 
swimmers,  and  they  rise  to  the  surface  of  the  water.  They  are  able  to 
live  in  sea-water  varying  in  concentration  from  Vie  m  to  6/s  m.  When  the 
larvae  are  put  into  a  pure  solution  of  NaCl+KCl  in  the  proportions  in 
which  these  two  salts  exist  in  sea-water,  they  will  all  fall  to  the  bottom 
of  the  vessel  which  contains  them.  They  are  unable  to  swim,  although 
they  may  live  for  a  number  of  hours  in  such  a  solution.  If  one  salt 
with  a  bivalent  cation  be  added,  for  example  CaCl2  or  Srds  in  sufficient 
quantity,  they  will  rise  to  the  surface  but  they  cannot  stay  there  very 
long.  If,  however,  enough  of  a  mixture  of  CaCl2+MgCl2  is  added,  in  the 
proportions  in  which  calcium  and  magnesium  are  present  in  sea-water, 
the  larvae  will  rise  to  the  surface  and  remain  there,  constantly  swimming. 
Various  concentrations  of  the  Na+K  mixture  were  employed  and  the 
concentration  of  bivalent  cations,  Mg+Ca,  required  to  preserve  motil- 


326         PROPERTIES  OF  THE  COLLOIDAL  CONSTITUENTS 

ity  in  each  solution  was  determined.  The  results  showed  that  the 
ratio  ^g  *^  was  constant  over  a  wide  diversity  of  concentrations. 
In  other  words  the  concentration  of  bivalent  ions  necessary  to  preserve 
motility  varied  directly  as  the  concentration  of  monovalent  ions  and 
not  as  the  square,  as  in  the  case  of  permeability.  While  motility, 
therefore,  is  affected  by  changes  in  permeability,  the  effects  upon 
motility  involve  changes  which  are  not  identical  with  those  which 
underlie  the  alterations  of  permeability. 

The  permeability  of  the  surface  of  the  cell  for  substances  dissolved  in 
water  is  presumably  determined  by  the  diameter  of  the  interstitial 
pores  filled  with  protein  jelly  which  comprise  the  spaces  between  the 
lipoidal  elements  of  the  superficial  emulsion.  We  have  seen  that 
permeability  is  affected  by  reagents  which  presumably  affect  the  solu- 
bility or  state  of  aggregation  of  the  protein  constituents  of  the  cell. 
We  should  expect,  however,  to  find  the  permeability  of  the  surface  of 
the  cell  also  affected  by  lipoid-solvents,  especially  if  these  enter  into  the 
lipoidal  droplets  and  increase  their  diameter. 


FIG.  18. — Showing  successive  effects  of  increasing  diameter  of  the  oil-droplets  in  an 
emulsion  upon  the  size  of  the  interstitial  spaces.  As  the  droplets  increase  in  size  until 
they  touch  each  other  the  area  of  the  interstitial  spaces  diminishes.  Further  increase 
in  the  diameter  of  the  oil-droplets  increases  the  sectional  area  of  the  interstitial  spaces. 

According  to  the  measurements  of  Osterhout,  the  various  lipoid- 
solvents,  in  particular,  ether,  chloroform,  chloral  hydrate  and  alcohol 
which  are  also  Anesthetics,  exert  two  effects  upon  protoplasm:  The 
one  consists  in  a  decrease  of  permeability  which  is  reversible,  i.  e., 
disappears  after  removal  of  the  anesthetic.  The  other  effect,  which 
requires  large  dosages,  in  an  increase  of  permeability  which  is  found 
to  be  irreversible.  Since  anesthesia  is  reversible  we  may  presume  it  to 
be  associated  with  the  former  of  these  effects  while  the  ultimate  toxic 
or  lethal  effects  of  these  drugs  may  be  referred  to  the  irreversible  in- 
crease of  permeability. 

The  absorption  of  these  substances  by  the  lipoidal  elements  of  the 
superficial  emulsion  with  consequent  increase  in  the  volume  of  the 
lipoidal  droplets  might  lead  either  to  decreased  or  increased  perme- 
ability for  substances  which  are  soluble  in  water.  Provided  the  lipoidal 
droplets  are  not,  in  the  normal  superficies  of  the  egg,  in  physical  con- 
tact with  one  another,  the  interstitial  spaces  between  the  droplets  will 


ORIGIN  OF  ACID  SECRETIONS  327 

be  reduced  in  diameter  by  the  swelling  of  the  droplets.  As  soon  as  the 
droplets  come  to  touch  one  another,  however,  any  further  increase  in 
their  diameter  will  push  their  peripheries  further  apart  and  increase 
the  diameter  of  the  interstitial  pores. 

This  will  be  clear  from  the  accompanying  diagram  depicting  the 
three  conditions  indicated  (Fig.  18).  It  can  readily  be  seen,  therefore, 
how  a  lipoid-solvent  may,  in  small  doses,  decrease  the  permeability  of 
cells  for  water-soluble  substances  and,  in  larger  doses,  increase  it. 

THE  ORIGIN  OF  ACID  SECRETIONS. 

It  has  always  been,  until  within  very  recent  years,  a  fact  exceedingly 
puzzling  to  physiologists  that  certain  secretory  glands,  particularly 
the  glands  of  the  gastric  mucosa  and  the  "salivary"  glands  of  car- 
nivorous molluscs,  elaborate  a  strongly  acid  secretion  from  an  alkaline 
medium,  namely  blood  or  other  tissue-fluids.  The  alkalinity  of  the 
medium  was,  of  course,  greatly  overestimated  by  the  earlier  observers. 
On  the  other  hand,  however,  the  results  of  the  most  exact  measure- 
ments show  that  the  blood  and  tissue-fluids  are  on  the  alkaline  side  of 
neutrality,  while  the  acidity  of  gastric  juice,  of  which  the  components 
must  in  the  long  run  have  been  derived  from  the  blood,  is  comparable 
with  that  of  a  hundredth-molecular  solution  of  hydrochloric  acid. 

The  first  hypothesis  which  was  advanced  in  explanation  of  this 
phenomenon  is  usually  but  erroneously  attributed  to  the  German 
biological  chemist,  Maly,  who  published  it  in  1874.  It  actually 
originated  with  an  American,  E.  N.  Horsford  whose  account  of  this 
hypothesis  is  contained  in  an  article  contributed  to  the  Proceedings  of 
the  Royal  Society  of  London  in  1869.  He  observed  that  if  a  mixture 
of  neutral  or  even  weakly  alkaline  salts,  such  as  the  Phosphates,  be 
enclosed  within  a  parchment-membrane  and  allowed  to  diffuse  through 
it  into  distilled  water,  the  water  outside  the  membrane  becomes  acid 
in  reaction,  while  that  within  the  membrane  becomes  correspondingly 
more  alkaline.  This  phenomenon  is  due  to  the  fact  that  the  diffusion- 
velocity  of  acids  is  more  rapid  than  that  of  the  alkaline  salts  which 
are  formed  within  the  .dialyzer.  From  the  alkaline  blood,  containing 
chlorides  and  phosphates,  therefore,  the  acid  hydrochloric  juice  was 
supposed  to  arise  in  an  analogous  manner.  The  difficulty  which  con- 
fronts this  hypothesis  is,  however,  that  it  proves  too  much,  since  by 
parity  of  reasoning  all  the  secretions  of  the  tissues  should  be  acid  in 
reaction,  whereas,  as  a  matter  of  fact,  the  majority  of  the  secretions 
resemble  the  blood  in  reaction  or  else,  as  in  the  case  of  the  pancreatic 
juice,  are  actually  more  alkaline  than  the  blood.  Moreover  the  effects 
observed  in  the  dialysis  of  salt  mixtures  are  too  small  in  magnitude  to 
account  for  the  relatively  high  acidity  of  gastric  juice.  An  alternative 
hypothesis  advanced  by  Koeppe  is  even  more  difficult  of  acceptance. 
This  investigator  supposes  that  the  gastric  mucosa  is  permeable  to 
sodium  ions  but  not  for  chlorine  ions.  As  sodium  ions  in  the  food 


328         PROPERTIES  OF  THE  COLLOIDAL  CONSTITUENTS 

leave  the  stomach  and  penetrate  the  tissues  an  equivalent  number  of 
hydrogen  ions  migrate  from  the  tissues  into  the  lumen  of  the  stomach 
and  there  combine  with  the  chlorine  ions  to  form  Hydrochloric  Acid. 
In  the  first  place  the  assumption  of  the  differential  permeability  of 
the  stomach-wall  for  sodium  and  hydrogen  ions  is  purely  gratuitous 
and  has  no  foundation  in  direct  observation,  and  in  the  second  place 
the  theory  would  require  the  presence  of  food  in  the  stomach  before 
acid  gastric  juice  could  be  secreted,  whereas,  as  Pavlov  has  shown,  the 
secretion  of  acid  gastric  juice  may  be  excited  reflexly  without  the 
presence  of  any  foodstuffs  in  the  stomach. 

T.  B.  Osborne  has,  however,  drawn  attention  to  a  mechanism  where- 
by an  acid  fluid  may  be  elaborated  through  the  intermediation  of 
Proteins.  When  Edestin  is  dissolved  in  sodium  chloride  solutions  and 
then  precipitated  by  passing  in  a  stream  of  carbon  dioxide,  it  is  found 
that  the  precipitate  contains  an  excess  of  combined  hydrochloric 
acid,  while,  on  the  other  hand,  an  equivalent  mass  of  sodium  carbonate 
or  bicarbonate  has  been  formed  in  the  fluid  and  may  be  estimated  by 
titration  with  methyl  orange.  When,  in  other  words,  the  excess  of 
sodium  hydroxide  is  neutralized  by  carbon  dioxide,  this  protein  com- 
pound of  sodium  chloride  breaks  up,  setting  free  sodium  hydroxide 
and  retaining  hydrochloric  acid  in  combination.  A  precisely  similar 
phenomenon  occurs  when  Red  Blood-corpuscles  are  repeatedly  washed 
with  isotonic  salt  solution  until  the  washings  become  perfectly  neutral 
and  are  then  suspended  in  neutral  sodium  chloride  solution  and  treated 
with  a  stream  of  carbon  dioxide.  The  external  fluid  becomes  alkaline 
and  the  blood-corpuscles  become  richer  in  chlorine  (Giirber).  In  this 
way  hydrochloric  acid  is  brought  into  combination  with  a  non-diffusible 
base,  and  may  be  subsequently  separated  from  it  by  hydrolytic  dis- 
sociation, followed  by  the  diffusion  of  the  hydrochloric  acid  into  the 
surrounding  medium,  or  in  the  particular  instance  under  consideration, 
into  the  gastric  juice.  We  may  infer,  therefore,  that  the  secretion  of 
an  acid  juice  depends  upon  the  existence  in  the  secreting  cells  of  a 
protein  which  is  capable  of  decomposing  sodium  chloride  in  the  presence 
of  carbon  dioxide.  The  appearance  of  the  free  hydrochloric  acid  in 
the  secretion  being  attributable  to  the  colloidal,  indiffusible  character 
of  the  protein  base. 


THE  SELECTIVE  ACTION  OF  TISSUES  AND  THE  "  OLIGODYNAMIC  " 
ACTIONS  OF  HEAVY  METALS. 

It  is  a  universal  phenomenon  in  living  tissues  that  despite  the  fact 
that  the  exact  composition  of  the  inorganic  milieu  is  so  definitely  related 
to  their  welfare  and  can  depart  so  little  from  normality  without  induc- 
ing disturbances  of  permeability,  yet  the  relative  proportions  of  the 
various  inorganic  constituents  of  the  protoplasm  do  not  conform  at 
all  to  -.the  proportions  subsisting  in  the  medium  which  they  inhabit. 


SELECTIVE  ACTION  OF  TISSUES  329 

Thus  the  Red  Blood-corpuscles  and  the  Skeletal  Muscles,  although 
bathed  by  fluids  which  contain  a  marked  excess  of  sodium  over  potas- 
sium salts,  nevertheless,  in  themselves,  contain  a  very  marked  excess 
of  potassium  over  sodium  salts.  Again,  although  in  fresh-water  streams 
the  relative  content  of  potassium  is  often  extremely  low,  the  plants 
which  live  in  them  are  capable  of  storing  up  a  comparatively  large 
amount  of  potassium  in  their  tissues.  One  of  the  most  extreme  instances 
of  this  selection  by  living  tissues  of  components  in  disproportion  to 
their  abundance  in  the  surrounding  medium  is  that  afforded  by  the 
presence  of  Iodine  in  considerable  amounts  in  the  tissues  of  the  Thyroid 
Gland  in  mammals  and  in  the  tissues  of  Marine  Algae.  Iodine  is  present 
in  normal  blood  only  in  undetectable  traces  and  in  sea-water  in  extra- 
ordinarily small  amounts. 

If  we  place  within  a  dialyzer  an  excess  of  diffusible  potassium  salts 
over  diffusible  sodium  salts  and  dialyze  against  a  solution  containing 
excess  of  diffusible  sodium  salts,  the  proportions  of  sodium  to  potassium 
within  and  without  the  dialyzer  sooner  or  later  readjust  themselves, 
approaching  equality.  Now  the  surface  of  the  living  cell,  although, 
perhaps,  sparingly  permeable  to  water-soluble  substances  is  neverthe- 
less not  absolutely  impermeable  to  them,  and  in  the  course  of  time  if 
the  inorganic  constituents  of  the  cell  are  present  therein  wholly  in 
diffusible  forms,  the  concentrations  of  the  various  inorganic  components 
within  and  without  the  cell  must  ultimately  attain  equality.  Even 
the  One-sided  Permeability  of  the  cell-surface  would  not  alter  the 
proportions  of  the  various  constituents  from  those  prevailing  in  the 
external  medium,  although  their  total  concentration  would,  in  conse- 
quence of  this,  be  constantly  maintained  at  a  somewhat  higher  level 
than  that  prevailing  in  the  external  medium.  Hence  this  phenomenon 
admits,  as  Loeb  has  pointed  out,  of  only  one-  explanation,  namely  that 
the  inorganic  constituents  of  a  tissue  which  are  found  therein  in  excess 
of  the  proportion  in  which  they  occur  in  the  fluids  which  bathe  it, 
must  exist  within  the  tissue  in  the  form  of  non-dissociated  and  non- 
diffusible  compounds.  "If  a  tissue  utilizes  one  kind  of  metal  in  this 
way,  for  example  K,  while  another  metal,  for  example  Na,  is  chiefly 
used  for  the  formation  of  dissociable  compounds  with  Na  as  the  free 
ion,  the  consequence  will  be  that  the  ashes  of  the  tissue  contain  K  and 
Na  in  altogether  different  proportions  from  those  in  which  they  are 
contained  in  the  surrounding  solution.  I  think  we  may  take  it  for 
granted  that,  at  least,  potassium  forms  a  non-dissociable  constituent 
of  the  protoplasm  of  a  number  of  tissues  of  animals  and  plants' ' 
(Loeb.) 

The  proteins  are  the  only  abundant  constituents  of  protoplasm 
which  possesses  the  amphoteric  property  necessary  for  simultaneous 
combination  with  acid  and  basic  radicals.  We  have  seen,  furthermore, 
that  the  compounds  of  proteins  with  inorganic  bases,  acids  and  salts, 
do  not  yield  any  inorganic  ions  to  the  solution;  they  are  non-dis- 
sociable compounds  in  so  far  as  the  inorganic  component  is  concerned. 


330        PROPERTIES  OF  THE  COLLOIDAL  CONSTITUENTS 

It  is  to  the  protein  compounds  in  the  main  that  we  must  look,  therefore, 
for  the  origin  of  the  selective  ability  of  tissues. 

Many  of  the  Heavy  Metal  Salts,  such  as  those  of  mercury,  silver, 
lead  or  copper  are  highly  toxic  for  living  organisms  in  extraordinarily 
high  dilutions.  Even  water  distilled  from  a  metallic  still,  or  collected 
in  a  metallic  condenser  may  be  extremely  toxic  to  many  forms  of  life. 
This  phenomenon  appeared  so  impressive  to  the  botanist  Nageli  that 
he  invented  a  special  phrase  "  oligodynamic  action"  to  describe  it. 

The  phenomenon  is  not  so  surprising  as  it  might  appear,  however, 
when  we  recollect  that  heavy  metal  ions  are  protein  precipitants  and 
especially  tend  to  form  insoluble  and  non-dissociated  compounds 
with  proteins.  The  effect  of  this  is  to  reduce  the  concentration  of 
heavy  metal  ions  in  any  region  containing  protein,  and  if  the  protein 
is  surrounded  by  a  medium  which  still  contains  free  metal  ions  these 
will  diffuse  in  to  take  the  place  of  those  precipitated  or  rendered  non- 
dissociable.  These  in  turn  will  be  removed  from  the  solution  and  so 
the  process  will  go  on  until,  although  the  original  concentration  of  metal 
ions  in  the  external  medium  may  have  been  very  small,  in  the  end  the 
concentration  of  combined  metal  in  a  cell  may  be  considerably  greater 
and  quite  sufficient  to  constitute  a  lethal  dosage.  As  W.  A.  Osborne 
has  shown,  this  sequence  of  events  may  be  directly  observed  by  plac- 
ing a  protein  solution  inside  a  parchment-dialyzer  and  immersing  the 
dialyzer  in  an  exceedingly  dilute  solution  of  mercuric  chloride.  The 
mercury  quickly  attains  a  higher  concentration  within  the  dialyzer 
than  without,  because  as  rapidly  as  it  enters  it  is  bound,  and  the 
osmotic  gradient  remains  positive  from  the  medium  without  to  the 
protein  solution  within  the  dialyzer. 

THE  BIOLOGICAL  INDIVIDUALITY  OF  TISSUES  AND  TISSUE- 
FLUIDS. 

In  discussing  the  various  compounds  which  the  Proteins  are  capable 
of  forming  we  had  occasion  in  Chapter  VIII  to  dwell  upon  the  existence 
and  the  properties  of  the  compounds  of  proteins  with  other  proteins 
and  especially  upon  the  demonstration  afforded  by  the  investigations 
of  Hardy,  that  the  Serum-globulin  which  is  separable  from  blood- 
serum  by  dilution  and  acidification  is  not  present  as  such  in  the 
untreated  serum  but  in  the  form  of  a  complex,  probably  arising  out  of 
the  union  of  several  protein  molecules. 

The  presence  of  these  protein  complexes  in  the  tissues  and  tissue- 
fluids  affords  a  simple  and  readily  intelligible  explanation  of  what 
would  otherwise  constitute  an  exceedingly  puzzling  fact,  namely,  the 
Biological  Individuality  of  the  various  tissues  and  tissue-fluids.  The 
individual  proteins  which  are  found  in  the  tissue-fluids  of  tolerably 
nearly  related  animals,  for  example  in  the  tissue-fluids  of  the  various 
species  of  mammalia,  appear,  on  analysis,  to  be  either  identical  or 


BIOLOGICAL  INDIVIDUALITY  OF  TISSUES  331 

very  nearly  identical  with  one  another.  Thus  the  casein  of  human 
milk  has  been  shown  by  Abderhalden  to  be  chemically  identical  with 
the  casein  of  goat's  milk,  in  so  far  as  the  relative  yields  of  the  various 
amino-acids  enable  us  to  judge.  Similarly  the  serum-albumins  and 
globulins  of  goose-blood  are  identical  with  those  of  horse-blood,  and 
the  investigations  of  Abderhalden  together  with  more  recent  analyses 
by  Gortner  and  Wuertz  have  shown  that  within  the  closest  approxi- 
mation attainable  by  present  methods  of  analysis  the  amino-acid 
yields  from  the  fibrins  of  ox-blood,  horse-blood,  sheeps'  blood  and  the 
blood  of  swine,  are  all  identical.  Yet  when  the  blood  or  blood- serum 
of  any  species  of  animal  is  injected  into  the  circulation  of  another 
species  it  is  treated  as  a  foreign  intrusion,  and  results  in  the  appear- 
ance of  specific  "antibodies"  such  as  the  Hemolysins  or  the  Precipitins 
which  react  with  the  blood  of  the  species  injected,  but  with  no 
other.  Thus  if  a  rabbit  be  injected  repeatedly  with  human  blood- 
serum,  the  serum  of  this  rabbit  acquires  the  abnormal  property  of 
causing  a  precipitate  to  form  when  it  is  mixed  with  human  serum. 
It  makes  no  difference  to  the  result  what  human  being  may  furnish 
the  serum,  but  if  we  employ  sera  from  other  and  unrelated  mammals 
we  obtain  little  if  any  precipitate  after  mixing  with  the  serum  of  the 
immunized  rabbit.  With  the  sera  of  related  species  some  precipitate 
will  be  obtained,  but  it  is  not  so  abundant  as  that  which  is  yielded  by 
human  serum.  The  relationship  of  man  to  the  primates  was  thus 
established  upon  a  quantative  basis  by  Nuttal,  to  whom  the  following 
measurements  are  due: 
Anti-human  serum  mixed  with : 

Blood  of :  Amount  of  precipit  ate.         Percentage. 

Man 0.031  100 

Chimpanzee 0.040  1301 

Gorilla 0.021  64 

Ourang 0.013  42 

Dog 0.001  3 

Cat         0.001  3 

Tiger 0.0005  2 

Ox 0.003  10 

Sheep 0.003  10 

Guinea-pig .      .  0.000  0 

Rabbit  ..; 0.000  0 

Kangaroo  (Macropus  bennetti) 0 . 000  0 

In  a  similar  manner,  if  a  rabbit  be  immunized  against  the  serum  of 
some  other  vertebrate  than  man,  the  serum  of  the  rabbit  so  treated 
will  develop  a  precipitin  for  that  species  and  its  near  relatives,  and  not 
for  other  vertebrates.  The  blood-serum  of  each  species,  and  in  fact  the 
tissues  and  tissue-fluids  in  general  of  each  species  are  so  many  separate 
Antigens,  producing  in  immunized  animals  antibodies  which  may  in 
certain  cases  be  related  to  one  another  but  which  are  clearly  not  in 

1  The  estimate  of  the  quantity  of  precipitate  yielded  by  chimpanzee-serum  was 
much  too  high,  because,  as  occasionally  happens,  the  precipitate  did  not  settle  properly 
and  its  true  value  could  not  be  estimated. 


332         PROPERTIES  OF  THE  COLLOIDAL  CONSTITUENTS 

any  case  identical  with  one  another.  Now  as  far  as  our  experience  ex- 
tends, all  antigenic  substances  are  proteins.  All  attempts  to  demonstrate 
antigenic  properties  in  substances  unrelated  to  proteins  have  resulted 
in  failure  and  in  particular  the  investigations  of  Fitzgerald  and  Leathes, 
have  shown  that  the  Lipoids  are  non-antigenic.  Yet,  as  we  have  seen 
the  individual  proteins  which  may  be  isolated  from  the  tissue-fluids  are 
identical  in  widely  differing  species. 

If,  however,  the  individual  proteins  which  are  separable  from  blood- 
serum  by  chemical  procedures  are  present  wholly  or  in  part  in  the 
unaltered  serum  in  the  form  of  complexes  of  several  proteins  united 
together,  we  can  readily  understand  how  different  sera  come  to  contain 
differing  antigens.  Two  protein-complexes  of  this  type  might  well 
be  built  up  out  of  identical  units,  and  yet  differ  fundamentally,  owing 
to  differences  in  the  combining  proportions,  and  consequently  in  the 
mode  of  linkage  of  these  units.  Just  as  a  wide  and  conceivably  infinite 
variety  of  proteins  may  be  built  up  out  of  differing  permutations 
and  combinations  of  eighteen  or  nineteen  amino-acids,  so  an  infinite 
variety  of  protein  complexes  might  be  built  up  by  the  union  in  varying 
proportions  and  arrangements  of  the  comparatively  limited  number  of 
different  proteins  which  are  individually  separable  from  a  tissue-fluid. 

In  pursuance  of  this  idea  Gay  and  Robertson  and  C  L.  A.  Schmidt 
have  investigated  the  antigenic  properties  of  several  compound  pro- 
teins. If  compound  proteins  differ  in  their  biological  specificity  from 
their  constituents,  then  a  Compound  Protein  should  represent  a  new 
Antigen  giving  rise  to  antibodies  for  itself,  as  distinguished  from  the 
antibodies  for  its  constituents.  Unfortunately  a  formidable  technical 
difficulty  stands  in  the  way  of  clearly  recognizing  the  presence  of  anti- 
bodies which  are  specific  for  the  compound  protein.  This  is  the  diffi- 
culty which  is  constituted  by  the  fact  that  any  protein  which  is  capable 
of  being  split  by  hydrolysis  into  moieties  which  are  still  proteins  (in 
the  sense  that  they  are  antigenic)  gives  rise  on  injection  into  animals 
to  antibodies  not  only  for  itself  but  also  for  these  split-products. 
Analogously,  a  compound  protein  gives  rise  to  antibodies  for  its  con- 
stituent parts,  and  it  is  only  possible  to  distinguish  between  these, 
which  would  appear  in  the  blood  of  immunized  animals  after  injection 
of  the  separate  constituents,  and  any  antibodies  which  may  be  formed 
for  the  compound  as  a  whole,  in  those  doubtless  exceptional  instances 
in  which  the  antibody  for  the  compound  reacts  with  a  constituent 
which  is  not  normally  antigenic. 

The  above-mentioned  observers  have  therefore  investigated,  from 
this  point  of  view,  certain  compound  proteins  in  which  one  constituent 
is  non-antigenic,  such  as  Protamine  Caseinate,  of  which  the  protamine 
constituent  is  non-antigenic  and  toxic,  while  the  casein  constituent  is 
antigenic  and  non-toxic,  and  Globin  Caseinate  of  which  the  globin  con- 
stituent is  toxic  and  non-antigenic. 

Protamine  caseinate  displays   no   antigenic   characteristics   which 


BIOLOGICAL  INDIVIDUALITY  OF  TISSUES  333 

enable  it  to  be  distinguished  from  casein.  It  is  non-toxic,  but  whether 
this  lack  of  toxicity  is  attributable  to  the  masking  of  the  toxic  proper- 
ties of  protamine  by  its  combination  with  casein,  or  to  the  smallness 
of  the  proportion  of  protamine  contained  in  the  compound,  has  not  yet 
been  definitely  established.  It  gives  rise  to  antibodies  for  Casein  by 
virtue  of  its  casein-content,  just  as  casein  gives  rise  to  antibodies  for 
its  infraprotein  split-product  Paranuclein,  but  it  does  not  give  rise  to 
any  antibody  which  will  react  with  its  protamine  constituent.  Globin 
caseinate,  however,  differs  very  markedly  in  its  antigenic  behavior 
from  either  of  its  constituents.  In  the  first  place  it  is  non-toxic,  and 
the  failure  to  exhibit  toxicity  can  hardly  be  attributable  to  dilution  of 
the  globin  constituent  by  admixture  with  casein  since  globin  caseinate 
contains  66  per  cent,  of  globin  (see  Chapter  VIII).  Still  more  striking 
is  the  fact  that  it  yields  antibodies  which  react  (i.  e.,  display  Alexin- 
fixation)  not  only  with  the  casein  constituent  of  the  compound  but  also 
with  the  globin  constituent.  It  would  appear  evident,  therefore,  that 
injection  of  globin  caseinate  into  animals  gives  rise  to  an  antibody 
which  does  not  appear  in  response  to  separate  injections  of  its  con- 
stituents. We  have  in  this  case  therefore  an  instance  created  in  labora- 
tory glassware  of  what  we  have  assumed  to  occur  in  tissue-fluids, 
namely  the  formation  of  a  protein  complex  which  differs  from  other 
proteins,  even  from  those  out  of  which  it  is  itself  built  up,  in  the  anti- 
bodies to  which  it  gives  rise  when  it  is  injected  into  animals. 

REFERENCES. 
GENERAL: 

Loeb:     Studies  in  General   Physiology,  Chicago,   1905.     The  Dynamics  of  Living 

Matter,  New  York,  1906      The  Mechanistic  Conception  of  Life,  Chicago,  1912. 

The  Organism  as  a  Whole,  New  York,  1916. 

Robertson:     Ergeb.  der  Physiol.,  1910,  10,  p.  216  (consult  for  literature). 
EFFECTS  OF  REMOVAL  OF  CALCIUM: 

Macallum:     On  the   Mechanism  of  the  Physiological  Action  of   the   Cathartics, 

University  of  California  Pubs.,  Physiology,  1906. 

Meltzer  and  Auer:    Am.  Jour.  Physiol.,  1905,  14,  p,  366;  1908,  21,  p.  400. 
Merckx:     Arch.  Int.  Pharmaco-dynamie,  1906,  16,  p.  301. 
Bancroft:     Pfliiger's  Arch.,  1908,  122,  p.  616. 

Hertz,  Cook  and  Schlesinger:     Proc.  Roy.  Soc.  Med.,  London,  1908,  2,  p.  23. 
Robertson  and  Burnett:     Jour.  Pharm.  Exp.  Ther.,  1911-12,  3,  p.  635. 
ANTAGONISTIC  ACTION  OF  SALTS  AND  BALANCED  SOLUTIONS: 

Loeb:     Am.  Jour.  Physiol.,  1899-1900,  3,  p.  434.     Proc.  Nat.  Ac.  Sc.,  Washington, 

U.  S.  A.,  1915,  1,  p.  473.     Jour.  Biol.  Chem.,  1915,  23,  p.  423. 
Osterhout:     Bot.  Gazette,  1906,  42,  p.  127;  1907,  44,  p.  259;  1908,  45,  p.  117;  1908, 

46,  p.  53;  1913,  55,  p.  446;  1914,  58,  pp.  178,  272,  367;  1915,  59,  pp.  242,  317; 

1915,  60,  p.  228.     University  of  California  Publications,  Botany,  1907,  2,  p    317; 

Jour.  Biol.  Chem.,  1905-06,  1,  p.  363;   1914,  19,  pp.  335,  493,  517;  1918,  34,  p. 

363.     Zeit.  Physikal.  Chem.,  1909,  70,  p.   408.     Science  N.  S.,  1911,  34,  p.    187; 

1912,  35,  pp.   Ill  and  112;  1912,  36,  pp.  350  and  637;  1913,  37,  p.  Ill;  1914,  40, 

p.   214;  1916,  44,  pp.  318,  395;  1917,  45,  p.  97.     The  Plant  World,  1913,  16,  p. 

129.     Jahrb.  wiss    Botan.,  1914,  54,  p.  645.     Proc.  Am.  Phil.  Soc.,  1916,  55,  p. 

533. 

Lillie:     Am.  Jour.  Physiol.,  1912,  30,  p.  1. 
Lipman:     Bot.  Gazette,  1909,  48,  p.  105;  1910,  49,  pp.  41  and  207.     Centr.  Bakt. 

Par.  und  Infek.:  1912,  32,  p.  58;  1912,  33,  p.  305;  1912,  35,  p.  647. 
Waynick:     Univ.  California  Pubs.,  Agric.  Sc. ,  1918,  3,  p.  135. 


334         PROPERTIES  OF  THE  COLLOIDAL  CONSTITUENTS 

ORIGIN  or  ACID  SECRETIONS: 

Osborne:    Am.  Jour.  Physiol.,  1901,  5,  p.  180. 
BIOLOGICAL  INDIVIDUALITY: 

Nuttall:     Blood  Immunity  and  Blood  Relationship.     London,   1904. 

Robertson:     Univ.  California  Pubs.,  Physiology,  1911,  4,  p.  25.    The  Physical  Chem- 
istry of  the  Proteins,  New  York,  1918.  • 

Gay  and  Robertson:     Jour.  Exp.  Med.,  1912,  16,  pp.  470  and  479;  1913,  17,  p.  535. ' 

Schmidt,  C.  L.  A.:     Univ.  California  Pubs.,  Pathology,  1916,  2,  p.  157. 

Gartner  and  Wuertz:     Jour.  Am.  Chem.  Soc.,  1917;  39,  p.  2239. 


PAET  III. 

THE  CHEMICAL  CORRELATION  OF  THE 

TISSUES. 


CHAPTER  XV. 

THE  VEHICLES  OF  CHEMICAL  CORRELATION;  BLOOD 
AND  LYMPH. 

THE  COMPOSITION  OF  THE  BLOOD. 

The  distributing  agents  which  accomplish  the  transportation  of 
substances  from  one  part  of  the  body  to  another  are  the  Blood  and 
Lymph.  Through  their  intermediation  oxygen  and  the  products  arising 
from  the  digestion  of  the  foodstuffs  are  carried  to  the  tissues,  the  waste- 
products  which  result  from  their  activity  are  carried  from  the  tissues 
to  the  excretory  organs,  and  an  exchange  of  products  between  diverse 
and  widely  separated  tissues  is  also  rendered  possible.  Among  this 
latter  class  of  materials  there  are  included  a  number  of  substances 
which,  arising  in  one  tissue  or  group  of  tissues,  stimulate  other  and 
distant  tissues  to  correlated  activity.  These  substances  are  collectively 
designated  Hormones,  or  chemical  messengers  (from  op/mco,  I  arouse, 
or  excite). 

The  blood  consists  of  a  suspension  of  cellular  elements,  the  red  cor- 
puscles or  Erythrocytes  and  the  white  corpuscles  or  Leukocytes  in  a  pale, 
straw-colored  or  almost  colorless  fluid,  the  Plasma.  Of  the  two  types 
of  corpuscles  the  erythrocytes  are  much  more  abundant  than  the 
leukocytes,  the  normal  average  number  of  erythrocytes  in  man  lying 
between  five  and  six  million  per  cubic  millimeter  of  blood,  while  the 
leukocytes  vary  in  number  between  7000  and  15,000  per  cubic  milli- 
meter. In  other  species  the  number  of  formed  elements  per  cubic 
millimeter  of  the  blood  may  te  higher  or  lower  than  in  man.  Thus  in 
the  mouse  the  normal  erythrocyte-count  lies  between  ten  and  twelve 
million  per  cubic  millimeter. 

When  the  blood  is  shed  from  the  vessels  it  forms  within  a  few  minutes 
a  gelatinous  clot,  which  is  due  to  the  separation  from  the  plasma  of  an 
insoluble  protein  Fibrin.  On  standing,  the  clot  shrinks  or  undergoes 
Syneresis,  expressing  a  colorless  or  very  pale  yellowish  fluid,  rich  in 


336  VEHICLES  OF  CHEMICAL  CORRELATION 

protein  and  containing  in  fact  all  of  the  constituents  of  the  plasma  with 
the  exception  of  the  formed  elements  and  the  protein  Fibrinogen, 
from  which  the  fibrin  arose.  This  fluid  is  termed  the  Serum,  and  it 
may  be  obtained  in  greater  abundance  and  more  rapidly  by  removing 
the  fibrin  from  freshly  shed  blood  by  whipping  it  with  glass  rods  or  by 
shaking  it  up  with  beads.  The  fibrin  adheres  in  long  strings  to  the  rods 
or  beads  and  may  be  removed  with  them  from  the  fluid  which  is  now 
termed  Defibrinated  Blood.  From  this  the  corpuscles,  red  and  white, 
may  be  removed  by  centrifugalization,  the  supernatant  fluid  consisting 
of  serum. 

The  relative  volumes  of  the  plasma  and  corpuscles  may  be  deter- 
mined in  several  ways  of  which  the  most  accurate  is  probably  the 
method  devised  by  Hoppe-Seyler,  which  suffers  from  the  disadvantage, 
however,  of  being  somewhat  lengthy  and  tedious.  Defibrinated  blood 
is  employed  for  the  estimation,  the  removal  of  fibrin  from  the  whole 
blood  introducing  only  a  very  slight  error  which,  if  desired,  may  be 
separately  estimated.  Three  determinations  are  made,  namely: 
(a)  The  total  protein  including  hemoglobin  in  1000  grams  of  whole 
blood,  (b)  The  total  protein,  including  hemoglobin,  in  the  blood- 
corpuscles  derived  from  1000  grams  of  blood  by  centrifugalization 
followed  by  repeated  washing  with  isotonic  salt  solution,  until  the 
washings  are  free  from  protein,  (c)  The  total  proteins  in  1000  grams 
of  serum  free  from  corpuscles.  The  difference  between  (a)  and  (6) 
yields  the  proteins  in  the  serum  contained  in  1000  grams  of  blood, 
so  that  the  ratio  ~  yields  the  proportion  of  1000  grams  which  is 
constituted  by  the  serum  in  that  weight  of  whole  blood.  For  example, 
in  an  actual  estimation,  the  total  protein  in  a  kilogram  of  blood 
amounted  to  172.9  grams,  while  the  corpuscles  from  this  amount  of 
blood  contained  124.0  grams  of  protein.  The  serum  in  a  kilo- 
gram of  blood,  therefore,  contained  172.9  —  124.0  =  48.9  grams  of 
protein.  One  kilogram  of  serum,  however,  contained  72.5  grams 
of  protein.  Therefore  the  serum  in  a  kilogram  of  blood  comprised 
HT§  ths  of  a  kilogram  or  674.5  grams.  The  Serum  (or  plasma  as 
it  is  termed  before  the  fibrin  is  removed  from  the  blood)  therefore 
forms  about  two-thirds  of  the  whole  blood  and  the  corpuscles  one-third. 
This  proportion  is,  however,  subject  to  very  wide  variations.  The  blood- 
count  itself,  i.  e.,  the  number  of  corpuscles  contained  in  a  cubic  milli- 
meter of  whole  blood,  is  variable  and  in  conditions  of  Anemia  may  fall 
to  one-half  the  normal  value.  Then  the  volume  occupied  by  the  indi- 
vidual corpuscles  varies  with  the  osmotic  pressure  of  the  serum,  hyper- 
tonicity  involving  shrinkage  and  hypotonicity  involving  dilation  of 
the  corpuscles. 

The  Electrical  Conductivity  of  the  whole  blood  compared  with  that  of 
the  serum  derived  from  it  may  also  be  employed,  as  Stewart  has  shown, 
for  the  determination  of  the  relative  volumes  of  the  corpuscles  and 
serum.  The  results  yielded  by  this  and  by  other  methods  are  in 
substantial  agreement  with  those  furnished  by  the  method  of  Hoppe- 
Seyler. 


COMPOSITION  OF  THE  BLOOD  337 

In  addition  to  the  red  and  white  blood  corpuscles  certain  other 
minute  formed  elements  are  also  found  in  shed  blood,  namely  the  Blood- 
platelets.  They  are  only  from  one-fifth  to  one-third  of  the  diameter 
of  the  red  corpuscles  and  they  do  not  contain  nuclei.  There  has  been 
very  much  discussion  as  to  whether  they  exist  in  the  circulating  blood 
as  such,  or  are  not  artefacts  arising  out  of  the  shedding  of  the  blood. 
They  have  been  regarded  by  various  observers  as  preformed  constit- 
uents of  the  circulating  blood,  as  detritus  from  the  destruction  of 
leukocytes  and  as  protein  coagula  or  sphere  crystals,  which  appear  in 
the  blood  whenever  the  endothelium  of  the  bloodvessels  is  injured. 
They  have,  however,  been  observed  by  Osier  in  the  blood  contained 
in  the  freshly  excised  capillaries  of  the  mesentery,  so  that  injury  to  the 
bloodvessels,  or  shedding  of  the  blood  from  the  vessels  is  not  an 
essential  prerequisite  to  their  formation.  On  standing  in  shed  blood 
the  platelets  swell  and  finally  break  up  and  disappear  and  there  is  some 
.indication  that  those  agencies  which  prevent  the  disintegration  of  the 
platelets  also  hinder  the  Coagulation  of  the  blood.  They  appear  to 
consist  of  protein  with  a  very  high  admixture  of  a  phospholipin  which 
resembles  Lecithin. 

The  Specific  Gravity  of  the  blood  necessarily  varies  with  its  total 
dilution,  that  is,  with  the  amount  of  fluid  which  has  recently  been 
absorbed  from  the  intestine.  As  a  rule  it  remains  between  the  upper 
and  lower  limits  of  1.060  and  1.054,  averaging  1.058  in  males  and  a 
little  less  in  females.  In  newborn  infants  the  blood  has  a  higher 
specific  gravity,  about  1.066. 

The  Chemical  Composition  of  the  Blood  is  very  constant  in  certain 
respects  and  highly  variable  in  others.  Thus  we  have  seen  that  the 
reaction,  osmotic  pressure  and  relative  proportions  of  the  various 
inorganic  constituents  are  exceedingly  invariable.  The  concentrations 
of  proteins,  glucose,  cholesterol  and  so  forth  are,  on  the  contrary,  very 
variable.  The  following  analytical  data,  cited  after  Abderhalden, 
are  therefore  not  to  be  regarded  as  affording  fixed  criteria  of  the  com- 
position of  the  blood  in  the  different  species  enumerated,  but  simply 
as  indications  of  an  approximate  average  composition.  Furthermore 
the  estimations  of  the  inorganic  constituents  are,  as  Abderhalden 
points  out,  merely  of  comparative  value,  since  the  analytical  errors 
involved  in  the  estimations  were  high,  although  presumably  of  similar 
magnitude  in  each  of  the  types  of  blood  investigated. 

At  the  time  that  the  above  analyses  were  made  the  whole  of  the 
Glucose  in  the  blood  was  supposed  to  be  confined  to  the  plasma  (or 
serum).  It  has  since  been  ascertained  by  Rona  and  Masing,  however, 
that  the  glucose  in  the  blood  is  contained  partly  in  the  erythrocytes 
and  partly  in  the  plasma.  It  is  not,  however,  distributed  between  these 
two  elements  in  proportion  to  their  relative  volumes.  In  addition  to 
the  various  substances  enumerated  in  these  analyses,  it  must  also  be 
remembered  that  blood  contains  small  amounts  of  Amino-acids,  derived 
by  absorption  from  the  intestine,  and  of  waste  products  such  as  Ammo- 
nia and  Urea  derived  from  the  metabolic  activities  of  the  tissues. 
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COMPOSITION  OF  THE  BLOOD  339 

The  Proteins  of  blood-serum  consist  of  an  admixture  of  albumins 
and  globulins.  It  is  quite  uncertain  how  many  different  proteins  the 
blood-serum  (or  plasma)  may  contain,  but  certain  fractions  can  be 
readily  distinguished  from  one  another.  Among  the  globulins  the 
"Insoluble  Globulin"  or  "Euglobulin"  may  be  readily  separated  by 
simple  dilution  of  the  blood-serum  with  from  ten  to  twenty  volumes  of 
distilled  water,  followed  by  acidification  with  dilute  acetic  acid  or  with 
a  stream  of  carbon  dioxide.  The  same  fraction  separates  out  on  sub- 
mitting blood-serum  to  dialysis.  An  additional  globulin  fraction,  the 
so-called  Pseudoglobulin  remains  in  solution,  but  may  be  coagulated 
by  half-saturation  of  the  serum  with  ammonium  sulphate,  and  there 
are  indications  that  this  substance,  in  turn,  is  not  a  single  chemical 
individual  The  Albumin  fraction,  which  is  not  coagulable  by  half 
saturation  with  ammonium  sulphate,  may  also  not  improbably  consist 
of  a  mixture  of  proteins.  Thus  from  the  serum  of  the  horse,  but  only 
with  great  difficulty  from  other  sera,  a  Crystalline  Serum  Albumin  may 
be  obtained  by  first  removing  the  globulins  by  half-saturation  with 
ammonium  sulphate  and  then  adding  more  ammonium  sulphate  until 
coagulation  of  the  albumins  just  begins,  and  allowing  the  mixture  to 
stand  for  some  time.  Only  a  portion  of  the  albumin  is  deposited  in 
crystalline  form,  however,  and  we  are  uncertain  whether  the  portion 
which  does  not  crystallize  merely  represents  the  quantity  requisite  to 
saturate  the  liquid  with  crystallizable  albumin,  or  whether  it  represents 
a  different  protein. 

In  addition  to  the  albumins  and  globulins  the  blood  often  contains 
very  small  amounts  of  Proteose,  and  also  a  glucoprotein,  termed  sero- 
mucoid  which  yields  glucosamin  on  hydrolysis.  It  is  present  in  blood- 
serum  only  to  the  extent  of  from  0.2  to  0.9  parts  per  thousand. 

It  has  been  noted  by  a  large  number  of  investigators  that  the  relative 
proportion  of  globulins  to  albumins  in  the  blood-serum  may  present 
remarkable  abnormalities  in  persons  or  animals  afflicted  with  certain 
Infections.  Normally  the  globulins  are  always  less  abundant  than  the 
albumin  fractions,  so  that  the  ratio  globulin  albumin  is  always  less 
than  unity.  In  animals  or  human  beings  infected  with  Streptococcus 
or  Staphylococcus ,  however,  the  ratio  may  be  much  more  than  unity, 
the  globulins  in  some  instances  amounting  to  as  much  as  eighty  or 
ninety  per  cent,  of  the  total  proteins.  The  question  of  the  origin  of 
this  remarkable  change  is  of  course  one  which  is  of  great  importance  to 
our  understanding  of  the  mechanisms  by  which  the  organism  protects 
itself  against  infections,  more  particularly  since,  in  the  case  of  Diph- 
theria at  least,  the  Antitoxins  resulting  from  infection  or  immunization 
have  been  found  to  be  associated  with  the  globulin-fraction  of  the 
serum. 

The  older  analyses  aiming  at  the  solution  of  this  problem  were 
subject  to  very  great  errors  and  uncertainties,  because  of  the  compara- 
tively large  volumes  of  blood  which  were  required  for  a  single  analysis. 
The  proteins  were  coagulated  by  alcohol,  dried  and  weighed,  while 


340 


VEHICLES  OF  CHEMICAL  CORRELATION 


in  another  sample  the  globulins  were  removed  by  half-saturation  with 
ammonium  sulphate  and  the  resulting  solution  of  albumins  was  purified 
by  dialysis  and  its  protein  content  determined  by  a  nitrogen  estimation 
or  by  coagulating,  and  weighing  the  dried  coagulum.  These  processes 
were  tedious,  inaccurate  and  time-consuming,  and  the  large  quantity 
of  blood  required  necessitated  restriction  of  analyses  to  single  samples 
or  to  samples  taken  at  rare  intervals  from  the  same  animals. 

The  recent  Refractometric  Method  of  Robertson  removes  these  sources 
of  inaccuracy  and  permits  the  determination  of  the  "  non-proteins" 
(including  proteoses),  globulins,  albumins  and  total  proteins  in  a 
quantity  not  exceeding  one  and  one-half  cubic  centimeters  of  serum. 
Samples  of  this  volume  may  be  taken  several  times  in  a  day  from  the 
ear  of  a  rabbit  without  any  evident  disturbance  due  to  hemorrhage, 
and  hence  the  effects  of  various  procedures  and  administrations  may  be 
studied  by  comparing  the  composition  of  the  blood-serum  of  the  animals 
before  and  at  successive  intervals  after  the  experimental  condition  is 
inaugurated. 

The  following  are  average  results  obtained  by  the  refractometric 
method  with  various  species  of  mammals  and  birds. 


Per  cent,  of  total 

Species. 

Non-protein, 
per  cent. 

Globulin, 
per  cent. 

Albumin, 
per  cent. 

Total 

protein, 
per  cent. 

protein. 

Globulin. 

Albumin. 

Horse        .... 

1.65 

3.5 

4.0 

7.5 

47 

53 

Albino  rat      ... 

1.61 

1.7 

4.2 

5.9 

29 

71 

Ox  

1.34 

2.2 

5.0 

7.2 

31 

69 

Hog     

1.49 

2.8 

4.3 

7.1 

39 

61 

Sheep        .... 

1.30 

1.1 

5.2 

6.3 

18 

82 

Goat   

1.43 

1.6 

4.9 

6.5 

24 

76 

Cat      

1.87 

2.6 

5.1 

7.6 

34 

66 

Dog     

.    2.01 

1.3 

4.8 

6.1 

21 

79 

Guinea-pig     . 

1.28 

0.9 

4.7 

5.7 

16 

84 

Hen     

1.42 

1.1 

3.5 

4.6 

25 

75 

Duck  

2.77 

1.9 

3.2 

5.1 

38 

62 

It  must,  however,  be  recollected  that  the  normal  proportion  of 
globulin  to  albumin  is  subject  to  considerable  fluctuation,  not  only  in 
different  individuals  of  the  same  species,  but  from  time  to  time  in  the 
same  individual.  The  following  are  estimations  made  by  Rowe  upon 
sera  derived  from  eighteen  normal  persons: 

The  last  four  determinations  were  made  upon  samples  obtained 
from  the  same  individual  on  different  dates.  It  will  be  observed  that 
the  globulins  in  these  normal  individuals  never  exceeded  thirty-two 
per  cent,  of  the  total  proteins,  although  ranging  from  this  proportion 
down  to  sixteen  per  cent,  in  different  individuals.  On  the  other  hand 
seventeen  persons  with  Syphilis,  and  yielding  a  strongly  positive 
Wassermann  Reaction  gave  values  for  the  proportional  globulin  content 
ranging  from  26  to  49  per  cent.,  and  averaging  35.7  per  cent.  Eight 


COMPOSITION  OF  THE  BLOOD  341 

persons  with  Pneumonia  had  a  globulin-ratio  of  from  27  to  50  per  cent, 
and  averaging  40  per  cent.  Other  infections  showed  corresponding 
increases  in  the  proportion  of  globulins  to  total  proteins  in  the  serum. 
Cases  of  Nephritis  gave  a  high  proportion  of  globulin  (24  to  50  per  cent.) 
while  those  in  which  nephritis  was  associated  with  the  accumulation  of 
salts  and  urea  in  the  blood  had  also,  of  course,  a  high  non-protein 
content.  On  the  other  hand  a  series  of  patients  with  diabetes  gave 
normal  values  for  the  globulin-ratio  excepting  in  one  instance  in  which 
a  local  infection  was  also  present.  Individuals  afflicted  with  various 
types  of  anemia,  hyperthyroidism,  goiter,  hemophilia,  chronic  bron- 
chitis, pellagra,  obesity,  lead-poisoning,  chronic  gastro-intestinal  dis- 
orders and  neurasthenia  presented  normal  values  for  the  protein-ratio. 
Exceptionally  high  values  of  the  proportion  of  globulin  to  albumin  in 
the  blood-serum,  therefore,  are  associated  with  Infections  or  else  with 
Toxemias. 

Globulin  expressed 

Non-proteinSj  Total  proteins,    in  per  cent,  of  total 

Sample  No.  Age.  per  cent.  per  cent.  protein. 

1  ....  27  1.2  7.8  25 

2  .   ...  30  1.3  7.4  30 

3  ....  36  1.3  7.3  32 

4  .  ."..  .   .  21  1.3  7.7  26 

5  24          1.1          7.6          16 


6  ....  30 

7  .....  32 

8  ..-..  48 

9  .   ,   .   .  19 

10  .   .".  .   .  25 

11  .  ,  .   .   .  48 

12  28 


.2  7.4  32 

.1  8.0  28 

.2  7.9  27 

.2  8.2  27 

.3  7.7  26 

.2  7,3  30 

.3  6.8  29 


13  ....  23  1.2  7.4  24 

14  ....  19  1.25  6.5  29 

15  ....  48  1.25  6.7  31 

16  ....  25  1.3  7.5  21 

17  ..;'-.  26  1.3  6.7  25 

18  ....  29  1.3  6.8  21 

19  ....  26  1.3  7.5  20 

20  .....  26  1.3  8.2  21 

21  .   .   .   .  26  1.3  8.2  18 

22  26  1.1  7.9  24 


Averages  .   .   .   ,'...          1.24         7.5          25.5 

The  origin  of  the  rise  of  globulins  in  infections  is  still  to  be  sought. 
It  is  not  due  to,  or  directly  correlated  with  the  development  of  anti- 
bodies in  the  circulation,  because  as  C.  L.  A.  Schmidt  has  shown,  a 
high  degree  of  immunity  to  pure  proteins  may  be  induced  without  any 
rise  of  globulins.  It  is  not  due  to  the  Leukocytosis  or  increase  in  white 
blood  corpuscles  which  often  accompanies  infection,  because  Hurwitz 
and  Meyer  have  shown  that  the  leukocyte-count  and  the  globulin 
increases  do  not  in  any  degree  run  parallel  to  one  another,  while  C.  L. 
A.  Schmidt  has  shown  that  the  leukocyte-count  may  be  reduced  to 
one-half  the  normal  in  rabbits  by  the  administration  of  Benzole  without 
causing  any  significant  alteration  of  the  globulin-ratio.  It  is  not  due  to 
alterations  of  bodily  temperature,  because,  as  Hanson  and  McQuarrie 


342  VEHICLES  OF  CHEMICAL  CORRELATION 

have  shown,  the  previously  reported  rise  of  globulins  after  the  adminis- 
tration of  Antipyrin  was  due  to  analytical  errors,  and  does  not  occur; 
The  same  observers  have  also  shown  that  therapeutic  agents  which 
markedly  accelerate  or  retard  metabolism,  namely  Thyroid  Extract 
and  Sodium  Cacodylate  respectively,  are  devoid  of  influence  upon  the 
protein  quotient,  and  Hanson  has  also  shown  that  the  previously 
reported  effects  of  Starvation  were  due  to  individual  fluctuations  and 
that  if  a  sufficient  number  of  analyses  be  made  neither  starvation  nor 
heavy  feeding  is  found  to  affect  the  quotient  in  any  constant  manner. 
On  the  other  hand  Buck  has  shown  that  if  Ether  or  Chloroform  be  admin- 
istered for  very  prolonged  periods  to  animals,  so  that  Albuminuria 
begins  to  appear,  the  globulin  quotient  rises,  far  more  markedly  than 
could  be  accounted  for  by  an  escape  of  serum-albumin  into  the  urine. 
This  observation  may  possibly  indicate  that  the  true  source  of  the 
marked  alterations  in  the  globulin-quotient  which  occur  in  infections 
and  toxemias  resides  in  alterations  of  the  Permeability  of  the  tissue- 
cells.  No  further  evidence  bearing  upon  this  possibility  is  as  yet, 
however,  in  our  possession. 

THE  COAGULATION  OF  THE  BLOOD. 

One  of  the  most  remarkable  properties  of  the  blood  is  that  which  it 
possesses  of  clotting  or  coagulating  in  a  brief  period  after  its  issuance 
from  the  bloodvessels.  The  clot  which  is  formed  is  a  markedly  con- 
tractile one  and  if  it  is  loosened  from  the  sides  of  the  vessel  to  which  it 
otherwise  adheres,  the  clot,  with  its  entangled  blood-corpuscles,  shrinks 
away  toward  the  center  of  the  vessel,  expressing  a  clear  white  or  pale 
yellow  serum  as  it  recedes.  This  phenomenon  is  known  as  Syneresis. 
If  a  clot  be  cut  into  pieces  with  a  knife  or  rod,  the  pieces  retract  from 
one  another  and  round  up  into  separate  masses. 

A  number  of  different  agencies  are  capable  of  preventing  the  clotting 
of  blood  wrhen  it  is  shed,  thus  the  various  Calcium  Precipitants,  such  as 
oxalates,  citrates,  sulphates  and  so  forth  will,  if  added  in  sufficient 
amounts,  prevent  or  delay  the  coagulation  of  the  blood  and  in  fact  a 
common  way  of  preparing  incoagulable  blood  is  to  receive  the  blood 
directly  from  the  vessels  into  a  solution  of  sodium  or  ammonium 
oxalate.  Such  Oxalated  Blood  as  it  is  called,  remains  fluid  and  inco- 
agulable until,  and  unless  a  soluble  calcium  salt  be  added  to  it  in  suffi- 
cient amount  to  remove  all  of  the  calcium-precipitating  agent.  Accord- 
ing to  Sabbatini  there  are  minimal  and  maximal  concentrations  of 
Calcium  Chloride  below  and  above  which  coagulation  is  inhibited.  The 
upper  limit  is  a  0.162  molecular  solution,  the  lower  about  one  thou- 
sandth part  of  this  concentration.  Salts  which  do  not  actually  pre- 
cipitate calcium,  such  as  sodium  citrate,  prevent  coagulation  by  reduc- 
ing the  concentration  of  free  Calcium  Ions  below  the  necessary  minimal 
limit. 

Other  agencies  which  will  prevent  coagulation  are  certain  solutions 


CO AGVL AVION  OF  THE  BLOOD  343 

of  Peptones  or  Proteoses.  When  these  are  injected  into  the  circulation, 
in  a  very  brief  period  the  blood  which  is  drawn  from  the  vessels  is 
found  to  be  incoagulable.  The  Peptone-plasma  obtained  from  this 
blood  by  centrifugalization  may  be  induced  to  coagulate  by  the  mere 
addition  of  a  suspension  of  leukocytes  obtained  from  lymph,  or  by  the 
addition  of  calcium  chloride  in  excess  of  the  amount  already  present 
in  the  blood,  or  by  acidification  with  carbon  dioxide  or  acetic  acid. 
Wooldridge  has  also  drawn  attention  to  the  very  interesting  property 
possessed  by  some  proteins  which  are  probably  Phosphoglobulins, 
namely  that  of  inducing  Intravascular  Clotting  if  injected  into  the 
circulation  gradually  or  in  small  doses;  while  they  render  the  blood 
Incoagulable  if  they  are  injected  more  quickly  or  in  larger  doses.  The 
former  effect  Wooldridge  designated  the  Positive  Phase  of  the  action 
of  the  protein,  the  latter  he  termed  the  Negative  Phase.  It  is  an 
especially  remarkable  fact  that,  according  to  Pickering,  albino  rabbits, 
and  the  Norway  hare  when  in  its  albino  condition,  are  immune  from  these 
effects.  These  various  phenomena  have  not  yet  received  any  adequate 
interpretation. 

Another  agent  which  renders  blood  incoagulable  is  the  extract  of 
leeches'  heads,  known  as  Hirudin.  Certain  Snake  Venoms  induce  a  like 
effect. 

'The  clotting  of  the  blood  is  in  the  first  instance  due  to  the  trans- 
formation of  a  soluble  protein,  Fibrinogen,  into  an  insoluble  modifi- 
cation, Fibrin.  This  was  conclusively  shown  by  the  investigations  of 
A.  Schmidt  and  of  O.  Hammarsten.  If  the  plasma  obtained  from  blood 
be  mixed  with  an  equal  volume  of  a  saturated  solution  of  Sodium  Chlo- 
ride a  precipitate  or  coagulum  of  fibrinogen  is  produced  which  may  be 
washed  repeatedly  in  half-saturated  sodium  chloride  solution,  redis- 
solved  in  dilute  sodium  chloride,  reprecipitated  by  half-saturation 
with  sodium  chloride  and  again  redissolved.  This  solution  of  fibrinogen 
in  from  1.0  to  1.5  per  cent,  sodium  chloride  will  not  clot,  however  long 
it  may  be  allowed  to  stand.  In  order  to  induce  it  to  clot,  another 
substance  must  be  added  to  it,  to  which  the  name  Thrombin  has  been 
applied. 

Thrombin  may  be  obtained  from  freshly-formed  fibrin.  It  is  best 
prepared  from  the  strings  of  fibrin  which  are  obtained  by  whipping 
freshly-shed  blood;  these  are  washed  in  cold  water  with  constant  knead- 
ing until  all  of  the  Hemoglobin  has  been  removed.  The  fibrin  is  then 
squeezed  dry,  minced  with  scissors,  and  then  covered  with  an  eight 
per  cent,  sodium  chloride  solution,  which  does  not  dissolve  the  fibrin, 
but  extracts  the  thrombin  which  is  associated  with  it.  The  mixture  is 
placed  in  a  refrigerator  for  forty-eight  hours,  and  then  filtered  through 
cheesecloth.  A  few  drops  of  the  viscous  -filtrate,  added  to  ten  c.c. 
of  the  fibrinogen  solution,  cause  immediate  clotting,  without  the 
addition  of  any  calcium  salt.  On  the  other  hand,  thrombin  solution 
unmixed  with  fibrinogen  will  not  clot,  whether  calcium  salts  be  added 
to  it  or  not. 


344  VEHICLES  OF  CHEMICAL  CORRELATION 

Since  calcium  is  necessary  for  the  coagulation  of  freshly-shed  blood, 
it  might  seem  reasonable  to  suppose  that  the  thrombin  solution 
contains  combined  or  associated  calcium,  which  suffices  to  permit  the 
process  to  go  forward.  This  is,  however,  not  at  all  the  case,  for  throm- 
bin may  be  purified  by  dialysis  and  precipitation  with  Acetone,  and 
when  this  has  been  done  twice  the  thrombin  is  found  to  be  perfectly 
free  from  calcium. 

The  true  secret  of  the  essentiality  of  calcium  in  the  clotting  of 
recently  shed  blood  lies  in  the  fact  that  thrombin,  as  such,  is  absent 
from  the  circulating  blood,  and  from  oxalated  plasma.  Instead,  we 
have  a  mother-substance,  Prothrombin  which  is  converted  by  calcium 
salts  into  thrombin.  This  fact  may  be  shown  in  a  variety  of  ways, 
among  which  the  following  may  be  cited:  Wooldridge  showed  that 
if  peptone  plasma  be  cooled  for  some  time  to  zero  degrees  centigrade 
a  precipitate  of  minute  discoidal  particles  collects  at  the  bottom  of  the 
container.  They  resemble  very  greatly  the  Blood-platelets  and  may, 
indeed,  actually  be  identical  with  them.  When  these  are  removed  from 
the  plasma,  clotting  of  the  fluid  is  now  very  difficult  to  induce  by  the 
customary  agents,  by  carbon  dioxide,  calcium  chloride  and  so  forth. 
Wright  subsequently  showed  that  the  same  precipitate  occurs  in  oxa- 
lated plasma  and  Hammarsten  showed  that  its  removal  prevented  the 
subsequent  clotting  of  the  plasma  by  the  addition  of  sufficient  calcium 
chloride  to  precipitate  the  oxalate  and  furnish  a  favorable  excess  of 
calcium  ions.  If,  however,  this  precipitate  be  treated  with  lime  salts 
and  the  calcium  subsequently  removed  by  oxalates,  it  now  is  found  to 
contain  very  active  Thrombin  which  quickly  induces  coagulation  in 
Oxalated  Plasma.  A  portion  at  least  of  the  material  in  the  discoidal 
particles  was,  therefore,  converted  by  the  calcium  salts  into  thrombin. 
This  constituent  is  prothrombin. 

Another  method  of  preparing  prothrombin  is  that  which  has  been 
devised  by  Howell.  Oxalated  blood  is  centrifugalized  and  the  plasma 
is  heated  to  54°  Centigrade.  This  coagulates  the  fibrinogen.  The 
filtered  plasma  is  treated  with  Acetone,  and  the  precipitate  is  collected 
upon  a  filter  and  dried.  When  the  prothrombin  is  required  for  use  the 
filter  paper  is  cut  into  small  pieces  and  extracted  for  about  one  hour 
with  dilute  sodium  bicarbonate  solution.  This  solution  does  not  cause 
clotting  of  pure  fibrinogen  or  of  oxalated  plasma  unless  it  is  first  treated 
with  calcium  chloride  (0.2  per  cent.). 

The  circulating  blood  contains  prothrombin,  therefore,  and  it  also 
contains  calcium  salts,  and  the  question  necessarily  arises  why  the 
prothrombin  is  not  converted  into  thrombin  in  the  vessels,  thus 
leading  to  intravascular  coagulation?  The  reason  that  this  does  not 
occur  is  that  the  conversion  of  prothrombin  into  thrombin  requires 
not  only  the  presence  of-  calcium  salts  but  also  another  factor,  derivable 
from  tissue  extracts,  which  Morawitz  termed  Thrombokinase,  but  which 
has  recently  been  identified  by  Howell  as  a  phospholipin,  namely, 
Kephalin. 


COAGULATION  OF  THE  BLOOD  345 

The  prompt  clotting  which  occurs  when  normal  blood  is  shed  is  due 
to  something  which  is  added  to  the  blood  when  it  comes  into  contact 
with  the  lacerated  tissues  over  which  it  flows,  or  which  is  derived 
from  the  disintegration  of  the  leukocytes  or  platelets  in  the  shed  blood. 
This  can  be  shown  by  employing  the  blood  of  Birds  or  Amphibians  in 
which  the  white  corpuscles  do  not  disintegrate  so  readily  after  shedding 
as  they  do  in  the  blood  of  mammalia.  If  a  paraffined  cannula  be  intro- 
duced into  an  artery  of  a  bird  and  the  blood  be  collected  in  a  paraffined 
centrifugal  tube  and  directly  centrifugalized,  the  plasma  which  is 
obtained  either  does  not  clot  at  all,  or  only  very  slowly. 

The  plasma  of  birds'  blood  which  is  thus  obtained  may  be  induced 
to  clot  if  any  of  a  large  variety  of  tissue  extracts,  such  as  leukocyte 
extract,  or  extracts  of  the  brain,  testes  or  thymus  be  added  to  it.  The 
active  substance  is  soluble  in  ether  and  with  difficulty  soluble  in 
alcohol,  and  it  contains  phosphorus  and  nitrogen.  Howell,  and  Mc- 
Lean have  shown  that  pure  Kephalin,  prepared  from  brain  or  other 
tissues  has  the  same  power  of  inducing  coagulation  as  the  whole  tissue 
extract,  while  other  phospholipins,  lecithin,  cuorin  and  sphingomyelin 
are  devoid  of  activity.  The  activity  of  kephalin  is  dependent  upon 
the  presence  of  unsaturated  linkages,  for  hydrogenated  kephalin,  or 
kephalin  that  has  become  oxidized  by  exposure  to  the  air,  is  inactive. 
That  some  other  factor  besides  the  mere  presence  of  unsaturated 
linkages  determines  the  action  of  kephalin  is,  however,  evident  from 
the  fact  that  the  great  majority  of  the  phospholipins  which  are  devoid 
of  Thromboplastic  Action  also  contain  unsaturated  linkages. 

The  prothrombin  in  oxalated  birds'  plasma  is  not  converted  into 
thrombin  by  kephalin  unless  calcium  salts  are  also  present.  Evidently 
therefore,  both  of  these  factors  cooperate  in  the  transformation  of 
prothrombin  into  thrombin. 

Two  views  of  the  mode  of  action  of  thrombin  upon  Fibrinogen  have 
been  advanced:  The  older  view,  originally  proposed  by  A.  Schmidt, 
regarded  thrombin  as  an  enzyme  which  converted  fibrinogen  into 
fibrin  by  hydrolysis,  just  as  Casein  is  converted  by  rennet  into  Para- 
casein.  The  foundation  of  this  view  was  twofold:  In  the  first  place 
the  thrombin  in  the  plasma  or  serum  of  shed  blood  is  inactivated  by 
heating  to  60°  centigrade  for  a  few  minutes  and  the  majority  of  the 
enzymes  are  similarly  inactivated  at  a  like  temperature.  In  the  second 
place  very  small  quantities  of  thrombin  are  requisite  to  produce  rela- 
tively large  quantities  of  fibrin.  Howell,  however,  has  adduced  a 
number  of  facts  which  militate  against  this  view.  In  the  first  place 
the  apparent  thermolability  of  thrombin  is  due  to  the  presence  of  salts 
or  other  substances  in  the  plasma  or  serum,  and  pure  thrombin,  freed 
from  inorganic  salts  by  dialysis,  will  withstand  boiling  for  five  minutes 
or  more  without  total  loss  of  activity.  If  to  the  same  solution  0.5 
to  1  per  cent,  of  sodium  chloride  be  added,  boiling  for  one  minute 
inactivates  the  thrombin  completely.  Of  course  this  fact,  in  itself, 
does  not  prove  that  thrombin  is  not  an  enzyme  for,  as  we  have  seen, 


346  VEHICLES  OF  CHEMICAL  CORRELATION 

many  enzymes,  particularly  those  of  bacterial  origin,  are  known  which 
are  not  inactivated  by  heating  or  in  which  the  inactivation  is  tem- 
porary or  reversible.  Moreover  it  is  not  really  certain  that  any  pure 
enzymes  are  thermolabile,  since,  with  one  exception,  no  pure  enzymes 
have  ever  been  prepared.  The  one  exception  is  that  afforded  by  the 
Laccase  or  oxidizing  enzyme  of  Medicago  sativa,  which  has  been  shown 
by  Euler  to  be  a  mixture  of  calcium  salts  of  aliphatic  hydroxy-acids. 
A  synthetic  mixture  of  calcium  glycollate,  citrate,  malate  and  mes- 
oxalate  has  the  same  oxidizing  action  as  the  vegetable  enzyme  and  is 
unaffected  by  boiling. 

The  evidence  afforded  by  the  effects  of  heating  is  therefore  incon- 
clusive either  for  or  against  the  view  that  thrombin  is  an  enzyme. 
Much  more  decisive  is  the  quantitative  relationship  of  the  fibrin-yield 
to  the  thrombin  which  has  been  added  to  the  fibrinogen  solution.  The 
following  are  estimates  obtained  by  Howell: 

0.  05  mgm.  of  thrombin  yielded  10. 75  mgm.  of  fibrin. 
0.16      "  "  "       34.00      "  " 

0.25      "  "  "       36.80      " 

0.64      "  "  "       42.50      " 

Moreover,  a  submaximal  quantity  of  thrombin  acting  upon  a  solution 
of  fibrinogen  will  never  furnish  a  full  yield  of  fibrin,  no  matter  how  much 
time  is  permitted  for  the  reaction  to  take  place.  Evidently,  therefore, 
thrombin  enters  into  and  determines  the  final  equilibrium  which  is 
attained  and  its  action  cannot  be  purely  catalytic. 

The  action  of  thrombin  upon  fibrinogen  is  specific  in  the  sense  that 
no  other  protein  is  similarly  modified  by  thrombin,  but  it  is  indifferent 
whether  the  thrombin  and  the  fibrinogen  are  derived  from  the  same 
or  related  or  even  unrelated  species  of  animals.  Thus,  Howell  has 
found  that  the  fibrinogen  of  all  vertebrates  is  converted  into  fibrin  by 
thrombin  derived  from  pigs'  blood. 

There  remains  to  consider  the  part  which  is  played  by  the  various 
factors  which  contribute  to  the  formation  of  thrombin  from  pro- 
thrombin.  Reasoning  from  the  analogy  afforded  by  the  conversion  of 
Trypsinogen  into  Trypsin  by  the  Enterokinase  of  the  succus  entericus, 
Morawitz  supposed  that  the  conversion  of  prothrombin  into  thrombin 
by  tissue-extracts  was  attributable  to  an  enzyme  which  he  designated 
Thrombokinase.  A  fact  which  encouraged  this  view  is  that  if  tissue- 
extracts  be  heated  to  from  56°  to  60°  Centigrade,  they  lose  their  throm- 
boplastic  activity,  and  it  was  inferred  that,  like  the  majority  of  the 
enzymes,  the  thromboplastic  substance  was  thermolabile.  Kephalin, 
however,  which  is  very  active  in  promoting  the  conversion  of  pro- 
thrombin into  thrombin  does  not  lose  its  thromboplastic  powers  when 
it  is  heated.  The  solution  of  this  apparent  contradiction  has  been 
supplied  by  the  investigations  of  Howell,  who  has  shown  that  if  the 
coagulum  which  forms  when  tissue  extracts  are  heated  to  60°  be  ex- 
tracted with  ether,  the  dried  ether  extract  has  all  the  thromboplastic 


COAGULATION  OF  THE  BLOOD  347 

activity  of  the  original  unheated  fluid.  Evidently  the  kephalin  in 
tissue-extracts  is  carried  down  with  the  protein  coagulum,  either 
physically  adherent  to  it  or  else  chemically  combined  with  it. 

According  to  Howell,  the  activation  of  prothrombin  by  kephalin 
is  due  to  the  removal  from  the  plasma  of  an  inhibiting  substance, 
Antithrombin,  which  is  present  in  varying  amounts  in  the  blood  of  dif- 
ferent species  of  animals.  The  proof  for  the  existence  of  this  substance 
is  as  follows:  If  thrombin  in  a  quantity  known  to  be  sufficient  to 
rapidly  coagulate  a  given  amount  of  a  solution  of  fibrinogen  be  pre- 
viously incubated  for  about  fifteen  minutes  at  blood-temperature  with  a 
small  amount  of  fresh  plasma  or  of  plasma  freed  from  fibrinogen  by 
heating  to  54°  C.,  the  ability  of  the  thrombin  to  coagulate  the  fibrino- 
gen is  found  to  have  become  very  much  impaired.  If,  however,  the 
plasma  has  been  previously  treated  with  kephalin,  its  power  of  inacti- 
vating thrombin  is  lost  or  very  much  weakened.  This  may  be  illus- 
trated by  the  following  data,  furnished  by  Howell.  The  following 
mixtures  were  prepared: 

Mixture  A.    Fresh  pigs'  plasma  +  equal  volume  of  water. 

Mixture  B.     Fresh  pigs'  plasma  +  equal  volume  of  kephalin  solution. 

The  mixtures  were  allowed  to  stand  for  thirty  minutes  and  then 
heated  to  54°  C.  to  coagulate  the  fibrinogen.  The  filtrates  were  then 
tested  for  their  antithrombin-content  as  follows: 


Mixture  A,  1  drop  +  Thrombin  5  drops — Incubation  of  15  mins. 

+  Fibrinogen  10  drops  =  Partial  clot  in  65  mins. 
Mixture  A,  1  drop  +  Thrombin  4  drops — Incubation  of  15  mins. 

+  Fibrinogen  10  drops  =  No  clot  in  2  hours. 
Mixture  A,  1  drop  +  Thrombin  3  drops — Incubation  of  15  mins. 

-j-  Fibrinogen  10  drops  =  No  clot  in  2  hours. 
Mixture  A,  1  drop  +  Thrombin  2  drops — Incubation  of  15  mins. 

+  Fibrinogen  10  drops  =  No  clot  in  2  hours. 

II 

Mixture  B,  1  drop  +  Thrombin  5  drops — Incubation  of  15  mins. 

+  Fibrinogen  10  drops  =  Clot  in  5  to  10  mins. 
Mixture  B,  1  drop  +  Thrombin  4  drops — Incubation  of  15  mins. 

+  Fibrinogen  10  drops  =  Clot  in  5  to  10  mins. 
Mixture  B,  1  drop  -f  Thrombin  2  drops — Incubation  of  15  mins. 

+  Fibrinogen  10  drops  =  Clot  in  5  to  10  mins. 
Mixture  B,  1  drop  +  Thrombin  2  drops — Incubation  of  15  mins. 

+  Fibrinogen  10  drops  =  Clot  in  10  to  15  mins. 

It  will  be  seen  that  the  inhibitive  action  of  mixture  A  is  totally 
absent  in  mixture  B,  which  has  been  incubated  with  kephalin.  These 
results  have  found  important  surgical  applications,  both  for  controlling 
hemorrhages,  for  which  purpose  gauze  soaked  in  an  aqueous  solution 
(or  emulsion)  of  kephalin  is  employed,  and  for  the  treatment  of  ab- 
normal tendency  to  prolonged  hemorrhage  in  cases  of  Hemophilia, 
which  Howell  interprets  as  being  due  to  an  abnormal  content  of  anti- 
thrombin  in  the  blood  of  the  patients  who  exhibit  it. 

Hemophilia  is  a  hereditary  condition,  and   is  further  peculiar  in 


348  VEHICLES  OF  CHEMICAL  CORRELATION 

that  it  is  almost  invariably  displayed  only  by  the  males  of  the  hemo- 
philic  family,  while  the  hereditary  tendency  to  hemophilia  is  trans- 
mitted by  the  females.  This  peculiar  mode  of  inheritance  is  also 
encountered  in  hereditary  Color-blindness  and  in  certain  other  instances 
of  inherited  abnormality;  it  is  designated  Sex-linked  Inheritance. 

We  may  therefore  sum  up  the  processes  and  substances  concerned 
in  the  coagulation  of  the  blood  as  follows: 

The  circulating  plasma  contains: 

Fibrinogen     +     Prothrombin  +  Calcium  salts      +     Antithrombin 


Thrombin      +     Calcium  salts  Neutralized 

by  kephalin. 


Hereafter  unessential 

I 
Fibrin. 

Howell  believes  that  in  addition  to  antithrombin  properly  so  called, 
which  inhibits  the  action  of  thrombin  upon  fibrinogen,  the  circulating 
plasma  also  contains  an  Antiprothrombin  which  inhibits  the  conversion 
of  prothrombin  into  thrombin  by  calcium  salts  and  is,  like  anti- 
thrombin, neutralized  or  inactivated  by  kephalin. 

In  regard  to  the  chemical  nature  of  the  substances  which  take  part 
in  the  coagulation  of  the  blood,  Fibrinogen  is  a  globulin,  being  like 
other  globulins  coagulable  by  half-saturation  of  its  solution  with 
ammonium  sulphate,  but  differing  from  the  serum-globulins  in  being 
also  coagulable  by  half-saturation  of  its  solutions  with  sodium  chloride. 
It  is  not  known  in  what  chemical  respects  Fibrin  differs  from  fibrinogen, 
but  the  results  of  Howell  and  others  would  seem  to  render  very  prob- 
able the  view  that  fibrin  is  a  compound  of  fibrinogen  and  thrombin. 
The  jelly  which  is  formed  by  the  conversion  of  fibrinogen  into  fibrin 
in  the  blood  or  in  neutral  or  faintly  acid  salt  solutions  is  of  exceptional 
interest  because,  as  Schimmelbusch  and  Howell  have  shown,  it  consists 
of  an  interlacing  network  of  acicular  crystals  enclosing  an  interstitial 
fluid  (Fig.  19).  If,  however,  fibrinogen  be  clotted  in  alkaline  solution 
the  jelly,  viewed  under  the  microscope  or  ultra-microscope  appears 
to  be  structureless.  The  crystalline  jellies  display  the  characteristic 
tendency  of  clotted  blood  to  shrink  in  and  express  fluid,  whereas  the 
structureless  jellies  do  not. 

The  source  of  the  fibrinogen  of  the  blood  appears  to  be  in  the  Liver, 
since,  as  Whipple  has  shown,  conditions  associated  with  injury  to  or 
insufficiency  of  the  liver,  such  as  Phosphorus  or  Chloroform  poisoning 
or  hepatic  cirrhosis  lead  to  a  marked  diminution  of  the  fibrinogen 
content  of  the  blood. 


COAGULATION  OF  THE  BLOOD  349 

Thrombin  may  be  a  protein,  but  if  so  then  it  is  protein  of  unusual 
properties,  for  it  is  not  coagulable  by  heat,  and  repeated  extraction 
with  chloroform  appears  to  remove  it  from  its  solution  in  water.  On 
the  other  hand  it  yields  the  biuret-  and  Millon  reactions  and  all  of 
the  reactions  for  Tryptophane,  and  it  is  coagulable  by  half-saturation 
with  ammonium  sulphate.  Putrefaction  does  not  destroy  it  and  in 
fact  often  seems  to  increase  its  activity.  These  properties  appear  to 
indicate  that  thrombin  may  be  a  protein  split-product,  possibly  a 
proteose. 


FIG.  19. — Fibrin  crystals  viewed  under  the  ultramicroscope.     (After  Howell.) 

The  nature  of  Antithrombin  is  unknown,  in  plasma  it  is  thermolabile 
while  the  antithrombin  in  leech  extracts  (Hirudin)  is  not.  It  is  uncer- 
tain, however,  whether  this  thermolability  may  not  be  due  to  asso- 
ciated impurities,  as  it  is  in  the  case  of  thrombin.  On  the  other  hand 
Antiprothrombin  appears  to  be  a  phospholipin,  McLean  having  shown 
that  Cuorin  from  heart-muscle  and  a  phospholipin  resembling  Jecorin 


350  VEHICLES  OF  CHEMICAL  CORRELATION 

from  the  liver  possess  marked  ability  to  inhibit  coagulation,  the  origin 
of  the  inhibition  being  the  delaying  or  prevention  of  the  formation  of 
thrombin  from  prothrombin  by  calcium  salts. 

THE  CHEMISTRY  OF  HEMOGLOBIN. 

The  red  coloring-matter  in  the  erythrocytes  of  the  vertebrates  is 
hemoglobin,  a  compound  protein  which  is  split  by  hydroylsis  into 
a  histone-like  protein,  Globin,  and  an  iron-containing  organic  acid, 
Hematin.  By  reason  of  the  power  which  it  possesses  of  forming  a  readily 
dissociable  compound  with  Oxygen,  hemoglobin  accomplishes  the  trans- 
portation of  oxygen  from  the  lungs  to  all  the  tissues  of  the  body. 
Other  pigments  fulfilling  a  like  function  are  found  widely  dispersed 
among  invertebrate  animals.  Thus  in  the  Arachnids  Crustacea  and 
Mollusca  there  is  found  a  protein  containing  copper,  which  has  been 
termed  hemocyanin  and  which  becomes  blue  when  saturated  with 
oxygen,  and  colorless  when  the  oxygen  is  liberated  again. 

The  content  of  Iron  in  hemoglobin  is  identical  in  all  species  of  animals. 
The  following  figures,  for  example,  are  given  by  Jaquet: 

Hemoglobin  from 

the  blood  of:  Per  cent,  of  iron. 

Dog 0.0336 

Horse     .      .      .      .      i      .      .      .  -  .      .      .    ".      ......  0.0335 

Ox ;-.....  ...  0.0336 

Hen .      .      .  :  .      .;:..      .      .      .  0.0335 

Assuming  that  each  molecule  of  hemoglobin  contains  one  atom  of 
iron,  this  implies  a  molecular  weight  for  hemoglobin  of  16,669  while 
complete  analyses  indicate  an  empirical  formula  approximating  to  the 
following: 

C759Hl208N2loS2FeO204 

If  we  examine  the  Absorption-spectrum  of  well  aerated  or  arterial  blood 
or  of  a  pure  solution  of  hemoglobin  which  has  been  shaken  with  air 
or  oxygen,  we  find  that  the  transmitted  light  contains  two  well-marked 
absorption-bands,  lying  between  the  Fraunhofer  lines  D  and  E.  The 
band  nearest  to  D,  termed  the  a  band  is  narrower,  but  darker  and 
sharper  than  the  ft  band  lying  nearer  to  E.  On  dilution,  the  &  band 
is  the  first  to  disappear.  On  concentration  the  bands  become  broader 
and  finally  appear  to  coalesce.  The  center  of  the  a  band  corresponds 
to  the  wave-length  X  =  579,  that  of  the  ft  band  to  the  wave-length 
X  =  542.  In  the  Photographic  Spectrum  a  band  may  also  be  seen  in  the 
ultraviolet  region,  near  to  G,  having  its  center  at  the  wave-length 
X  =  415.  This  band,  which  was  first  detected  by  Soret,  has  been 
proposed  as  a  means  of  detecting  hemoglobin  in  high  dilutions,  since 
it  is  still  perceptible  in  solutions  containing  only  one  part  of  hemoglobin 
in  40,000,  while  the  bands  in  the  visible  spectrum  are  no  longer  per- 
ceptible at  a  dilution  of  one  in  fifteen  thousand.  The  absorption-band 


CHEMISTRY  OF  HEMOGLOBIN  351 

in  the  ultraviolet  spectrum  is,  however,  not  characteristic  of  hemo- 
globin. It  is  also  shown  by  solutions  of  its  protein  component,  Globin, 
and  more  or  less  distinctly  by  solutions  of  many  other  proteins.  It  is 
distinctly  visible  in  the  light  transmitted  through  solutions  of  Tyrosine, 
Phenylalanine  and  other  aromatic  ammo-acids,  to  which  radicals  its 
presence  in  the  protein  absorption-spectrum  is  due. 

It  was  first  shown  by  the  English  physicist,  Stokes,  that  if  blood  be 
placed  under  a  vacuum,  or  acted  upon  with  a  reducing-agent  such  as 
an  alkaline  solution  of  ferrous  sulphate  or  ferrous  tartrate  (known 
as  Stokes'  Reagent),  or  warm  solutions  of  the  alkaline  sulphides,  the 
absorption-spectrum  of  the  solution  changes.  Only  one  band  is  now  to 
be  seen  in  the  visible  spectrum,  where  formerly  there  were  two.  This  lies 
between  D  and  E,  nearer  to  D  than  to  E.  The  same  spectrum  is  sup- 
plied by  the  blood  of  asphyxiated  animals.  This  absorption-spectrum 
is  due  to  hemoglobin  as  distinguished  from  the  Oxyhemoglobin  which 
is  formed  when  hemoglobin  solutions  are  aerated.  The  center  of  the 
band  lies  at  wave-length  X  =  559.  The  band  in  the  photographic 
spectrum  is  at  the  same  time  shifted,  as  Gamgee  has  shown,  the  center 
of  this  band  in  solutions  of  Reduced  Hemoglobin  lying  nearer  the  visible 
spectrum  than  it  does  in  solutions  of  oxyhemoglobin.  The  color  of 
solutions  of  oxyhemoglobin  is  the  typical  scarlet  of  arterial  blood; 
solutions  of  reduced  hemoglobin  are  darker,  with  a  slightly  purple 
hue  and  they  also  exhibit  the  phenomenon  of  Dichroism,  the  color  of 
light  reflected  from  the  surface  of  the  solution  being  green,  while 
transmitted  light,  as  we  have  stated  is  red,  with  a  slightly  purple  tinge. 

By  the  action  of  oxidiz ing-agents  reduced  hemoglobin  is  rapidly 
converted  into  oxyhemoglobin,  but  the  further  action  of  many  oxidiz- 
ing-agents  such  as  ozone,  potassium  permanganate,  potassium  ferri- 
cyanide  and  chlorates  results  in  the  formation  of  a  modification  of 
oxyhemoglobin  which  is  designated  Methemoglobin.  The  absorption 
spectrum  of  methemoglobin  resembles  that  of  oxyhemoglobin,  except- 
ing that  the  /3  band  is  more  intense  than  the  a  band  and  a  third  band  is 
present  between  C  and  D.  The  color  of  methemoglobin  solutions 
is  chocolate-brown,  changing  to  red  when  the  solution  is  rendered  acid, 
the  absorption-spectrum  changing  at  the  same  time  and  showing  only 
one  absorption-band  between  C  and  D.  The  oxygen-content  of  met- 
hemoglobin appears  to  be  identical  with  that  of  oxyhemoglobin,  but 
it  is  much  more  firmly  combined  and  is  not  given  up  under  a  vacuum, 
nor  is  it  displaced  by  a  stream  of  Carbon  Monoxide.  When,  however, 
methemoglobin  is  treated  with  Stokes'  reagent  reduced  hemoglobin 
is  reformed  and  this  in  turn  forms  oxyhemoglobin  on  shaking  the 
solution  up  with  air.  Methemoglobin  is  often  spontaneously  formed 
when  arterial  blood  is  allowed  to  stand  in  sealed  tubes  and  it  may 
be  found  in  transudates  and  cystic  fluids  stained  with  blood,  or  in  old 
extravasations  of  blood  following  upon  injuries. 

The  blood  of  animals  which  have  been  asphyxiated  by  illuminating 
gas  is  of  a  peculiar  florid  cherry-red  color,  which  does  not  change  when 


352  VEHICLES  OF  CHEMICAL  CORRELATION 

Stokes'  reagent  is  added  to  it.  This  is  due  to  the  presence  of  Carbon 
Monoxide  Hemoglobin,  which  may  also  be  obtained  by  blowing  a 
stream  of  carbon  monoxide  or  of  illuminating  gas  through  a  solution 
of  oxyhemoglobin  or  reduced  hemoglobin.  In  the  former  case  the 
oxygen  combined  with  the  hemoglobin  is  quantitatively  displaced  by 
the  carbon  monoxide,  a  given  volume  of  carbon  monoxide  displacing 
an  equal  volume  of  oxygen.  We  can  readily  distinguish  between 
normal  arterial  blood  and  the  blood  obtained  after  carbon  monoxide 
poisoning,  in  the  first  place  by  the  lack  of  effect  of  Stokes'  reagent 
upon  the  color  of  the  carbon  monoxide  hemoglobin,  and  in  the  second 
place  by  the  effect  of  adding  concentrated  sodium  hydroxide  (specific 
gravity  1.3)  in  the  proportion  of  two  volumes  of  sodium  hydroxide 
solution  to  one  volume  of  blood.  Blood  containing  carbon-monoxide 
hemoglobin  yields  a  cinnabar-red  precipitate,  whereas  normal  blood 
yields  a  dingy  brown  precipitate.  Furthermore,  Tannic  Acid  yields 
with  normal  blood  a  brownish-green  precipitate,  and  with  carbon- 
monoxide  blood  a  pale  crimson-red  precipitate.  The  spectrum  shows 
two  absorption-bands  similar  to  those  of  oxyhemoglobin  but  nearer 
to  the  violet  end  of  the  spectrum.  The  carbon  monoxide  may  be 
dissociated  from  the  hemoglobin  by  the  prolonged  action  of  a  vacuum 
or  of  a  stream  of  oxygen  or  an  indifferent  gas. 

The  quantity  of  oxygen  or  carbon  monoxide  which  combines  with 
one  gram  of  hemoglobin  is  1.34  c.c.  at  0°  C.  and  760  m.m.  Hg. 
This  corresponds  to  one  molecule  of  oxygen  or  carbon  monoxide  for 
every  atom  of  iron  in  the  hemoglobin  molecule.  If,  therefore,  we 
regard  the  molecule  of  hemoglobin  as  containing  one  atom  of  iron, 
the  reaction  between  hemoglobin  and  oxygen  appears  as  a  simple 
bimolecular  reaction  as  follows: 

Hb  +  O2  ^  HbO2 

The  reaction  proceeding  from  left  to  right  when  the  partial  pressure 
of  oxygen  is  increased,  as  it  is  in  the  lungs,  and  from  right  to  left  when 
the  partial  pressure  of  oxygen  is  reduced,  as  it  is  in  the  tissues.  Sim- 
ilarly the  interaction  with  carbon  monoxide  may  be  represented  as 
follows: 

Hb  +  CO  ^1  HbCO 

Designating  the  concentration  of  reduced  hemoglobin  in  any  solu- 
tion by  the  symbol  Cr,  that  of  oxyhemoglobin  by  the  symbol  Co,  and 
that  of  oxygen  by  the  symbol  "b,"  then  applying  the  mass-law  to  the 
balanced  reaction: 

Hb     +     O2    ^    HbO2 

Cr  b  Co 

We  would  have,  at  equilibrium: 

Cr   X  b   =  KCo 


CHEMISTRY  OF  HEMOGLOBIN  353 

where  "K"  is  a  constant  which  is  characteristic  of  the  equilibrium,  and 
represents  the  ratio  of  the  velocities  of  the  opposed  reactions.  The 
concentration  of  oxygen  in  the  solution  will,  of  course,  be  directly 
proportionate  to  the  partial  pressure  Po  of  oxygen  in  the  atmosphere 
above  the  solution  and  to  the  absorption-coefficient  at  of  oxygen  in 
water  at  the  particular  temperature  "t"  which  is  employed.  We 
therefore  have : 

b     =     Po.at 

and:. 

^    =  Kat  Po 

Hence  in  a  solution  of  hemoglobin  brought  into  equilibrium  at  any 
given  temperature  with  a  mixture  of  nitrogen  and  oxygen,  such  as  air, 
by  shaking  or  by  exposure  over  a  very  extensive  surface,  as  in  the 
capillaries  of  the  lungs,  the  ratio  of  oxyhemoglobin  to  reduced  hemo- 
globin should  be  directly  proportional  to  the  partial  pressure  of  oxygen 
in  the  atmosphere  to  which  it  is  exposed. 

This  relationship  was  investigated  by  C.  Bohr  who  found  so  many 
irregularities  which  were  apparently  inconsistent  with  the  equation 
that  he  inferred  the  existence  of  several  different  compounds  of  hemo- 
globin with  oxygen.  The  whole  question  was,  however,  reinvestigated 
by  J.  Barcroft  and  his  collaborators  with  greatly  improved  technique 
and  it  was  ascertained  that  the  irregularities  observed  by  Bohr  were 
due  to  inconstant  contamination  of  the  hemoglobin  by  crystalloids 
and  that  in  properly  dialyzed  solutions  the  relationship  deduced  from 
the  mass-law  holds  good  with  exactitude.  The  origin  of  the  irregulari- 
ties in  solutions  containing  inorganic  electrolytes  resides  in  the  ten- 
dency of  hemoglobin  to  polymerize  in  such  solutions,  Roaf  having 
found  that  while  hemoglobin  in  distilled  water  exerts  an  Osmotic 
Pressure  corresponding  to  a  molecular  weight  of  16,000,  in  sodium 
chloride  solution  the  osmotic  pressure  corresponds  to  a  molecular 
weight  of  32,000.  On  rendering  this  latter  solution  alkaline  the  molec- 
ular weight  of  the  hemoglobin  again  falls  to  16,000,  the  weight 
which  is  also  indicated  by  the  iron-  and  sulphur-contents,  assuming 
each  molecule  of  hemoglobin  to  contain  one  atom  of  iron. 

The  influence  of  Temperature  upon  the  Equilibrium-constant  of  a 
balanced  chemical  reaction  is  expressed  by  the  well-known  thermo- 
dynamical  equation: 

KT      =     KTo  •  e~T  ^f) 

where  KT  is  the  value  of  the  equilibrium-constant  at  the  temperature  T, 
KTO  is  the  value  of  the  constant  at  temperature  To,  "  q"  is  the  heat  given 
out  by  the  conversion  of  one  gram-molecule  of  the  substance  and 
"e"  is  the  base  of  the  natural  or  "Napierian"  logarithms.  The 
validity  of  this  equation  for  the  reaction  between  oxygen  and  hemo- 
23 


354  VEHICLES  OF  CHEMICAL  CORRELATION 

globin  has  also  been  established  by  Barcroft,  as  the  following  data 
reveal.  The  oxygen  pressure  was  constantly  maintained  at  10  mm.  Hg 

Percentage  of  hemoglobin  converted  into  oxyhemoglobin. 
At  16°  24°  32°  38°  49° 

Observed 92-  71  37  18  6 

Calculated 90  71  41  22  6 

from  which  figures  and  the  above  equation  it  is  easy  to  deduce  that 
"q"  or  the  heat  given  out  when  one  gram-molecule  of  hemoglobin 
unites  with  oxygen,  is  28,000  calories.  Now  the  heat  given  out  when 
one  gram  of  hemoglobin  unites  with  oxygen  is  1.85  calories.  Hence 
we  have  the  simple  ratios: 

weight  in  grams  of                    Heat  given  out  by  one  gram- 
one  gram-molecule  molecule 28, 000 

Weight  of  one  gram  Heat  given  out  by  one  gram  1.85 

whence  the  weight  in  grams  of  one  gram-molecule  of  hemoglobin  is 
calculated  to  be  15,200,  which  estimate,  when  one  recollects  the  number 
and  variety  of  measurements  which  enter  into  it,  is  in  extraordinarily 
good  accord  with  the  known  molecular  weight  of  hemoglobin,  namely, 
about  16,000. 


FIG.  20.— Hemin  crystals,  magnified.     (After  Preyer.) 

By  the  action  of  acids,  alkalies  or  heat  in  the  presence  of  oxygen, 
hemoglobin  can  readily  be  split  up  with  the  liberation  of  Hematin. 
If  this  hydrolysis  is  accomplished  in  the  presence  of  hydrochloric  acid 
the  substance  obtained  is  the  hydrochloride  of  hematin  or  Hemin, 
which  may  be  readily  recognized  by  its  characteristic  crystalline  form 
(see  Fig.  20). 

Alkaline  solutions  of  hematin  show  pronounced  Dichroism,  being 
red  in  thick,  and  green  in  thin  layers,  while  acid  solutions  of  hematin 
are  brown.  The  solid  substance  forms  glistening  bluish-black  amor- 
phous masses.  The  hydrochloride,  however,  is  brown. 

If  hematin  be  dissolved  in  strong  Sulphuric  Acid,  on  diluting  the 


CHEMISTRY  OF  HEMOGLOBIN 


355 


solution  a  dark  red  substance,  Hematoporphyrin,  or  iron-free  hematin 
is  deposited,  the  iron  originally  contained  in  the  hematin  molecule 
being  left  in  the  solution  in  the  form  of  Ferric  Sulphate.  Hemato- 
porphyrin is  identical  with  a  substance  known  as  Hematoidin  which 
is  frequently  found  in  the  form  of  microscopical  rhombic  crystals  in 
old  extravasations  of  blood  or  apoplectic  clots.  It  is  also  identical 
with  Bilirubin,  the  red  coloring-matter  of  the  bile. 

When  hemoglobin  is  decomposed  by  alkalies  in  the  absence  of  oxygen, 
we  obtain  Hemochromogen,  or  "reduced  hematin."  This  substance 
yields  bright  red  solutions  in  alkaline  media,  acids  very  quickly  change 
it  into  hernatoporphyrin  and  a  ferrous  salt: 


Hematin. 


2H2O   +  2HC1 


H2 


Hematoporphyrin. 


By  reduction  of  hematoporphyrin  we  obtain,  among  other   products, 
a  substance  known  as  Hemopyrrole,  C8Hi3N,  which  is  a  methyl  propyl 

pyrrole: 

HC  -  C  -  CH2.C2H5 


HC 


C— CH3 


NH 


From  its  quantitative  composition  and  the  abundance  of  Methyl 
Pyrrole  derivatives  among  its  decomposition-products,  it  appears 
probable  that  hematin  may  be  built  up  out  of  four  methyl  pyrrole 
radicals  united  by  iron  and  oxygen.  The  hydrochloride,  or  Hemin 
may  possibly  be  represented  by  the  following  structural  formula: 


-CH=C(OH)—  C=C— CH=CH— C C— CH3 


HC  CH 

\ 


O   FeCl 


\/ 
NH 


CH3— C 


HC 


::— CH=C(OH)—  < 


^— CH3 


\/ 
NH 


CH 

/ 


HC 


CH 


NH 


The  extensive  investigations  of  Marchlewski,  to  whom  we  owe 
much  of  our  knowledge  of  these  pigments,  have  resulted  in  establishing 
the  very  close  relationship  which  exists  between  hematin  and  Chloro- 
phyll, the  green  pigment  of  plants.  Thus  among  the  products  resulting 


356  VEHICLES  OF  CHEMICAL  CORRELATION 

from  the  decomposition  of  chlorophyll,  a  substance,  Phyloporphyrin, 
is  obtained  which  differs  from  hematoporphyrin  only  in  containing 
two  hydrogen  atoms  in  the  place  of  two  hydroxyl-groups.  The  attempt 
has  been  made  to  transform  hematoporphyrin  into  phyloporphyrin 
by  reduction,  but  this  attempt  has  as  yet  only  been  partially  successful, 
only  one  of  the  hydroxyl  groups  in  hematoporphyrin  having  been 
replaced  by  hydrogen. 

The  close  relationship  of  hematin  to  chlorophyll  at  once  suggests 
the  possibility  that  the  necessary  radicals  for  the  binding  of  hemo- 
globin may  be  obtained  by  animals  from  the  decomposition-products 
of  chlorophyll.  The  pyrrole  grouping  may  of  course  be  obtained  from 
the  Proline  and  Oxyproline  constituents  of  the  protein  molecule,  but  it  is 
a  question  whether  the  synthetic  activity  of  the  hemopoietic  tissue 
in  the  red  marrow  of  the  bones  goes  so  far  as  to  build  up  hsmatin 
from  pyrrole  or  whether,  rather,  somewhat  more  complex  fragments 
of  hematin  may  not  be  requisite.  It  is  true  that  chlorophyll  is  not 
digestible  by  the  hydrolytic  enzymes  of  our  alimentary  system,  but 
that  does  not  exclude  the  possibility  of  bacterial  digestion  in  the  lower 
intestine,  and  as  a  matter  of  fact,  Marchlewski  has  shown  that  chloro- 
phyll does  actually  in  part  disappear  when  introduced  into  the  aliment- 
ary canal  of  animals.  Abderhalden  has  suggested  that  the  failure  of 
inorganic-iron  therapy  in  certain  cases  of  Anemia  may  be  attributable 
to  lack  of  certain  decomposition-products  of  chlorophyll  in  the  diet, 
or  to  lack  of  the  proper  assimilation  or  utilization  of  these  products 
which  he  conceives,  may  be  necessary  for  the  synthesis  of  hemoglobin. 

THE  CRYSTALLINE  FORMS   OF  HEMOGLOBIN  IN   RELATION  TO 
THE  BIOLOGICAL  INDIVIDUALITY  OF  THE  BLOOD. 

The  constant  percentage  of  iron  in  the  hemoglobins  derived  from 
different  Vertebrata  invites,  but  does  not  establish  the  accuracy  of  the 
supposition  that  the  hemoglobins  from  different  sources  are  identical. 
While  the  quantitative  composition  of  hemoglobin  must  be  the  same 
in  all  species,  yet  there  exist  a  very  large  number  of  conceivable  ar- 
rangements of  the  various  radicals  and  groupings  in  the  molecule,  and 
of  stereochemical  differences  not  detectable  by  mere  analysis.  In  fact 
Reichert  and  Brown  have  in  recent  years  very  strongly  advocated 
the  view  that  the  hemoglobin  of  every  species  differs  chemically  or 
stereochemically  from  that  of  every  other,  basing  their  view  upon  the 
results  of  their  monumental  investigation  of  the  crystalline  forms  of 
hemoglobin  derived  from  different  sources. 

Crystals  of  hemoglobin  are  readily  obtained  from  the  blood  of  cer- 
tain animals  by  the  mere  evaporation  of  blood  "laked"  by  ether.  This 
procedure  suffices  in  the  case  of  the  blood  of  the  rat,  for  example.  In 
many  cases  it  is  necessary  to  cool  the  blood  to  zero  and  in  some  to  add 
alcohol  to  reduce  the  solubility  of  the  hemoglobin.  Generally  speaking 
the  best  method  to  induce  crystallization  is  to  add  from  one  to  five 


CRYSTALLINE  FORMS  OF  HEMOGLOBIN  357 

per  cent,  of  Ammonium  Oxalate  to  freshly  shed  blood,  which  not  only 
prevents  clotting  but  accelerates  the  process  of  crystallization,  then 
lake  the  corpuscles  by  shaking  up  the  blood  with  ether,  remove  the 
debris  of  corpuscles  by  centrifugalization  and  allow  the  fluid  to  evapo- 
rate on  a  microscopic  slide.  In  some  cases  the  nature  of  the  agent  em- 
ployed to  lake  the  blood  or  induce  Hemolysis  is  of  importance  in  deter- 
mining the  ease  of  crystallization.  Thus  if  dogs'  blood  be  laked  with 
Toluol,  an  abundance  of  crystals  of  hemoglobin  is  easily  obtained  by 
merely  cooling  the  laked  blood  in  a  refrigerator. 

The  results  of  Reichert  and  Brown  have  shown  that  the  crystals 
obtained  from  the  blood  of  different  species  are  never  identical  in 
form.  From  an  enormous  number  of  measurements  of  crystal-angles, 
etc.,  conducted  upon  hemoglobins  derived  from  a  very  wide  variety 
of  species  these  observers  conclude  that  the  crystals  of  the  different 
species  of  any  one  genus  belong  to  the  same  crystallographic  system 
and  generally  to  the  same  crystallographic  group,  and  they  have 
approximately  the  same  axial  ratios,  or  their  ratios  bear  a  simple 
relation  to  each  other.  In  other  words  the  hemoglobin  crystals  of 
any  genus  are  isomorphous,  but  not  identical.  In  some  cases  this 
Isomorphism  may  be  extended  to  include  several  genera,  but  this  is 
usually  not  the  case  unless,  as  in  the  case  of  the  dogs  and  foxes,  for 
example",  the  genera  are  very  closely  related.  On  the  other  hand  the 
oxyhemoglobin  obtained  from  the  same  species  always  crystallizes  in 
the  same  form,  although  often  with  a  different  "habit"  when  obtained 
by  different  methods  of  preparation.  But  upon  comparing  the  hemo- 
globins from  different  species  of  a  genus  it  is  always  found  that  they 
differ  from  one  another  to  a  greater  or  less  degree  in  angles  or  axial 
ratio,  in  optical  characters,  and  particularly  in  those  characters  com- 
prised under  the  general  term  "Crystal  Habit,"  so  that  one  species  can 
usually  be  distinguished  from  another  by  the  form  of  its  hemoglobin 
crystals  (Fig.  21). 

A  clear  relationship  is  thus  seen  to  subsist  between  the  physico- 
chemical  behavior  of  a  constituent  of  organisms,  and  their  place  in  the 
phylogenetic  scale  of  relationships  as  established  by  their  gross  mor- 
phology, and  a  long  stride  has  been  taken  toward  the  establishment  of 
a  physicochemical  basis  for  morphological  distinction.  The  further, 
and  entirely  independent  question  now  arises,  however,  as  to  the 
chemical  origin  of  the  observed  physicochemical  phenomena. 

Our  experience  with  the  crystallography  of  inorganic  and  the 
simpler  organic  substances  has  led  us  to  infer  with  a  considerable 
degree  of  confidence  that  substances  which  show  differences  in  crystal- 
lographic structure  are  different  chemical  substances.  Crystal  form 
is  affected  even  by  isomeric  modifications  which  analysis,  unaided  by 
other  methods  of  investigations,  fails  to  reveal.  Now  the  enormous 
number  of  atoms  in  a  protein  molecule  encourages,  at  first  sight,  the 
supposition  that  an  enormous  and  indeed,  for  all  practical  purposes, 
an  infinite  number  of  isomerides  are  possible  between  which  the  most 
refined  methods  of  analysis  would  not  enable  us  to  distinguish,  but 


358 


VEHICLES  OF  CHEMICAL  CORRELATION 


FIG.  21. — Oxyhemoglobin  crystals  of  various  animals.  1,  the  goose;  2,  the  Tasmanian 
devil  (Sarcophilus  ursinus) ;  3,  the  kangaroo  (Macropus  giganleus) ;  4.  the  horse ;  5 ,  the 
guinea-pig;  6,  the  long-armed  baboon  (Papio  langlceldi).  (After  Reichert  and  Brown.) 


CRYSTALLINE  FORMS  OF  HEMOGLOBIN  359 

which  would  very  probably  differ  from  one  another  in  the  morphology 
of  their  crystals.  In  point  of  fact,  however,  the  available  number  of 
isomers  would  be  very  greatly  restricted  by  the  necessity  of  maintain- 
ing unaffected  the  amino-acid  groupings  of  the  protein  moiety,  which 
could  not  differ  materially  in  different  species  without  leading  to  de- 
cided differences  in  the  chemical  behavior  of  the  hemoglobins,  which 
have  not  been  observed  by  any  investigator.  Further  doubt  is  thrown 
upon  this  interpretation  of  the  facts  by  the  observation  of  Hiifner, 
recently  confirmed  with  the  utmost  precision  by  Butterfield,  Heubner 
and  Rosenberg,  and  Schumm,  that  the  characteristic  Absorption- 
bands,  and  the  ratio  of  the  absorption  of  light  in  different  parts  of 
the  spectrum  of  hemoglobin  are  absolutely  identical  in  species  so  far 
removed  from  one  another  as  the  horse  and  man  (Schumm)  or  the 
rabbit,  sheep,  and  hog  (Heubner  and  Rosenberg).  Now  these  are 
properties  which  we  would  anticipate  might  be  materially  affected  by 
internal  differences  of  atomic  arrangement. 

Further  reason  for  doubting  the  correctness  of  referring  the  differ- 
ences of  crystal  structure  displayed  by  the  hemoglobins  of  different 
animals  to  internal  differences  in  the  molecule  of  the  hemoglobins  is 
supplied  by  the  observation  of  Loeb  and  Brown  that  the  crystal-form 
of  the  hemoglobin  of  the  mule  is  intermediate  in  character  between  that 
of  the  horse  and  that  of  the  donkey.  For  if  we  assume  that  each  different 
crystal-form  represents  a  different  internal  atomic  arrangement  of 
the  hemoglobin  molecule,  then  the  number  of  such  arrangements,  even 
if  very  great,  must  nevertheless  be  limited.  The  number  of  possible 
forms  of  crystals  must,  therefore,  also  be  limited,  and,  moreover,  the 
possible  modifications  of  forms  must  be  discontinuous,  i.  e.,  there  must 
exist  forms  between  which  no  intermediate  forms  are  possible.  This 
being  the  case  it  would  be  very  remarkable  indeed  were  the  hybridi- 
zation of  two  closely  related  species  to  lead  to  the  synthesis  of  a  new 
isomeric  variety  of  hemoglobin  not  yet  appropriated  by  any  existing 
species  of  animal  and,  in  addition,  lying  between  the  hemoglobins  of 
the  parent-species.  If  analogous  phenomena  should  be  displayed  by 
all  hybrids  and  by  all  varieties  and  mutations  that  might  have  arisen 
or  might  conceivably  arise  in  the  future,  we  would  have  to  admit  that 
the  hemoglobins  already  recognizable  as  differing  from  one  another  in 
crystalline  form  are  only  a  small  proportion  of  those  which  are  realisable. 

A  much  more  reasonable  supposition  is  that  embodied  in  the  view 
that  the  differences  in  crystal-form  observed  by  Reichert  and  Brown 
were  attributable,  not  to  the  internal  variation  of  atomic  grouping  in 
the  hemoglobin  molecules,  but  to  external  variations  in  the  milieu 
from  which  they  are  crystallized.  The  technique  adopted  by  Reichert 
and  Brown  was  to  induce  crystallization  directly  in  the  laked  blood. 
Now  we  know  from  the  observations  of  the  immunologists  that  the 
blood-plasma  from  any  species  of  animal  differs  antigenically  from 
that  derived  from  any  other  species,  and  since  all  known  antigens  are 
proteins,  we  infer  that  the  proteins  or,  more  probably,  the  compound 
Protein  Complexes  in  blood-plasmas  derived  from  different  species  are  in 


360  VEHICLES  OF  CHEMICAL  CORRELATION 

certain  definite  respects  different  from  each  other.  The  crystals  of  each 
species  studied  by  Reichert  and  Brown  were  therefore  deposited  from  a 
different  medium,  and  it  is  not  improbable  that  the  observed  differ- 
ences between  the  crystals  are  attributable  to  these  known  differences 
in  the  media  in  which  they  were  formed.  It  is  well  known  that  crystal- 
habit  is  modified  by  alterations  of  the  medium  from  which  the  crystals 
are  deposited.  That  modifications  of  this  origin,  so  great  as  to  prevent 
inclusion  of  the  crystals  formed  in  different  media  in  the  same  iso- 
morphic  series,  have  not  hitherto  been  observed  in  the  domain  of  inor- 
ganic chemistry  is  not  improbably  attributable  to  the  simpler  character 
of  the  conditions  accompanying  crystallization  in  inorganic  or  non- 
colloidal  media.  We  have  seen  that  there  are  many  reasons  for  sup- 
posing that  proteins,  even  in  solution,  are  disposed  in  a  certain  reticular 
structure  (cf.  Chapter  XIII),  and  if,  as  the  facts  which  we  dwelt  upon 
in  connection  with  the  properties  of  the  compounds  of  proteins  with 
each  other  would  seem  to  indicate,  characteristic  protein  complexes, 
formed  by  the  union  in  differing  proportions  of  a  relatively  small 
number  of  simpler  protein  components,  exist  in  each  type  of  blood- 
plasma,  we  may  well  suppose  that  the  reticular  structure  of  the  solu- 
tions comprising  these  plasma  would  likewise  differ  from  one  another. 
Having  regard  to  the  markedly  cohesive  properties  of  proteins,  crystal- 
lization within  the  meshes  of  such  a  reticulum  might  very  conceivably, 
through  external  strains  imposed  by  points  of  attachment  to  the  reticu- 
lum, modify  the  effects  of  the  internal  strains  which  find  their  expres- 
sion in  crystal  form. 

This  hypothesis  finds  decided  support  in  the  fact,  first  observed 
by  Halliburton,  and  confirmed  by  Reichert,  that  the  crystal  form  of 
oxyhemoglobin  derived  from  a  given  species  may  be  profoundly 
modified  by  admixture  with  the  blood  of  another  species.  The  follow- 
ing are  illustrative  results  obtained  by  Halliburton,  the  "normal" 
form  of  rat-hemoglobin  crystals  being  rhombic,  those  obtained  from 
guinea-pigs  being  normally  tetragonal,  and  those  from  squirrels'  blood 
hexagonal. 

Form  of  hemoglobin  crystals  deposited 
Blood  of  Mixed  with  that  of  from  the  mixture. 

Rat  Squirrel  Both  rhombic  prisms  and  hexagons  present. 

Rat  Guinea-pig  No  rhombic  prisms  of  the  shape  usually  seen 

in  rats'  blood  present;  no  tetrahedra; 
crystals  are  all  rhombic  prisms  with 
hexagonal  habit. 

Squirrel  Guinea-pig  Hexagonal     plates     and     retrahedra     both 

present;  many  tetrahedta  imperfect;  the 
tetrahedra  all  reduced  to  about  half  the 
si2e  of  those  prepared  from  the  unmixed 
blood  of  the  same  guinea-pigs. 
Squirrel  Fine  rhombic  needles  and  hexagonal  plates 

both  present  in  abundance. 

Guinea-pig  The  greater  number  of  the  crystals  formed 

are  very  small  tetrahedra  about  a  quarter 
the  size  of  those  prepared  from  the  blood 
of  the  same  guinea-pig.  The  optical 
properties  are,  however,  the  same;  rhom- 
bic prisms,  very  slender,  like  those  of 
dogs'  blood  are  also  seen. 


THE  CHEMICAL  DETECTION  OF  BLOOD  361 

According  to  Reichert,  the  degree  of  modification  of  crystal  form 
induced  by  admixture  of  two  bloods  depends  very  greatly  upon  the 
proportion  in  which  they  are  mixed. 

In  view  of  these  facts  there  can  be  little  doubt  that  the  nature  of 
the  milieu  in  which  crystallization  occurs  does  play  an  important  part 
in  determining  the  form  of  the  crystals  which  are  deposited,  and  having 
regard  to  the  known  individuality  of  the  plasma  from  different  bio- 
logical species,  it  would  appear  unnecessary  to  seek  further  for  the 
origin  of  the  differences  in  crystal  form  of  the  oxyhemoglobins  derived 
from  blood  of  different  species  of  animals. 

In  this  way  we  can  also  interpret  the  changes  in  crystal-form  which 
Halliburton  observed  to  result  from  repeated  Recrystallization  of 
hemoglobin,  for  as  Wichmann  and  more  recently  Katz  have  shown, 
the  crystalline  proteins  swell  in,  or  absorb  the  surrounding  fluid 
menstruum  in  a  manner  analogous  to  the  swelling  of  jellies.  A  number 
of  recrystallizations  are  therefore  required  to  remove  completely  traces 
of  the  original  menstruum  in  which  crystallization  occurred. 

Bradley  and  Sansum  believe  that  the  hemoglobins  from  different 
animals  are  antigenically  different,  because  guinea-pigs  sensitized  to 
ox-  or  dog-hemoglobin  failed  to  display  Anaphylactic  Shock,  or  reacted 
but  slightly  to  hemoglobins  of  other  origins,  while  they  reacted  strongly 
to  the  hemoglobin  with  which  they  were  sensitized.  As  the  hemo- 
globin preparations  employed  by  Bradley  and  Sansum  were  admittedly 
(with  the  exception,  they  believe,  of  dog-hemoglobin)  not  free  from 
contamination  by  serum,  the  interpretation  of  these  results  is  open  to 
serious  question.  Doubt  is  especially  thrown  upon  this  evidence  for 
the  specificity  of  hemoglobins  from  different  species  by  the  fact  that 
the  animals  sensitized  to  the  purest  preparation  of  hemoglobin  em- 
ployed, that  of  the  dog,  reacted  strongly,  not  only  to  dog-hemoglobin, 
but  also  to  dog-serum.  Observers  are  not  all  agreed  that  pure  hemo- 
globin is  antigenic;  its  protein  component,  globin,  certainly  is  not, 
and  having  regard  to  the  investigations  of  Wichmann  and  Kat, 
cited  above,  revealing  the  marked  ability  of  crystalline  proteins  to 
absorb  the  menstruum  from  which  they  are  deposited,  and  to  the 
observation  of  Schulz  and  Zsigmondy  that  Egg-albumin  must  be 
recrystallized  from  3  to  6  times  in  order  to  remove  appreciable  con- 
tamination by  other  proteins,  we  may  infer  that  in  all  probability 
the  specificities  demonstrated  by  Bradley  and  Sansum  are  serum- 
specificities  and  not  hemoglobin-specificities. 

THE  CHEMICAL  DETECTION  OF  BLOOD. 

The  chemical  detection  of  blood  and  identification  of  blood-stains 
is  often  of  the  very  gravest  medicolegal  import.  The  older  methods 
of  detection  depended  upon  microscopical  identification  of  blood- 
corpuscles,  and,  of  course,  a  very  slight  degree  of  putrefactive  change, 
or  the  drying  of  a  blood-stain  upon  a  garment  rendered  the  detection 


362  VEHICLES  OF  CHEMICAL  CORRELATION 

of  these  formed  elements  impossible.    This  was  succeeded  by  the  far 
more  delicate  and  reliable  Hemin  Test,  which  consists  in  placing  a  drop 
of  suspected  fluid  or  saline  extract  of  shreds  of  stained  fabrics,  upon 
a  microscope-slide,  adding  a  crystal  of  salt  and  a  drop  of  glacial  acid, 
heating  the  fluid  to  boiling  by  passing  the  slide  to  and  fro  over  a  small 
flame,  and  then  examining  the  fluid,  as  it  cools,  for  hemin  crystals. 
This  test  may  be  successfully  employed  with  samples  of  blood  far 
advanced  in  decomposition.  A  still  more  delicate  test,  however,  is  the 
Benzidine  reaction.    This  depends  upon  the  power  of  an  enzyme  or 
Peroxidase,1  which  is  present  in  blood,  to  decompose  Hydrogen  Peroxide, 
liberating  nascent  oxygen  which  oxidizes  the  benzidine  with  the  produc- 
tion of  a  green  or  blue  color.    Properly  conducted,  this  test  will  detect 
one  part  of  blood  in  three  hundred  thousand,  which  means,  in  effect,  that 
a  murderer  may  wash  his  blood-stained  hands  in  a  bath  full  of  water, 
and  yet  if  any  drainage  remains  unemptied  at  the  bottom  of  the  bath, 
the  fact  that  he  has  done  so  may  be  detected  with  certainty.    Never- 
theless even  more  delicate  tests  are  available.    TJius  Buckmaster  has 
found  that  if  an  alcoholic  solution  of  Guaiaconic  Acid  be  added  to  blood 
together  with  hydrogen  peroxide,  a  blue  color  may  be  produced  at  a 
dilution  of  one  in  five  million.   This  test  is  also  given  by  perfectly  fresh 
Milk  collected  and  bottled  with  aseptic  precautions,  but  it  is  not  given 
by  the  milk  which  is  ordinarily  obtainable  in  the  market. 

For  the  identification  of  the  Species  from  which  blood  is  derived  we 
rely  upon  the  antigenic  Specificity  of  blood.  The  suspected  fluid  is 
mixed  with  anti-human  serum  prepared  by  immunizing  a  rabbit 
against  human  blood.  The  mixture  is  incubated,  and  the  occurrence 
of  a  flocculent  precipitate  indicates  that  the  suspected  fluid  contained 
either  human  blood  or  the  blood  of  an  anthropoid  ape.  Since  "The 
Murders  in  the  Rue  Morgue"  must  be  admitted  to  have  constituted 
an  entirely  exceptional  problem,  the  alternative  thus  presented  does 
not  furnish  any  serious  basis  for  uncertainty. 

THE  ORIGIN  AND  COMPOSITION  OF  LYMPH. 

The  tissues  are  not,  excepting  in  a  very  few  situations,  bathed  by 
blood  itself,  but  by  the  Lymph,  which  is  derived  from  blood,  and 
through  the  intermediation  of  which  the  substances  dissolved  or 
combined  in  blood  are  brought  into  physical  contact  with  the  proto- 
plasm of  the  living  cells. 

There  was  formerly  much  discussion  of  the  question  whether  lymph 
is  elaborated  from  the  blood  by  a  process  of  active  secretion,  consti- 
tuting an  Exudate,  or  whether,  on  the  contrary,  it  is  a  Transudate, 
derived  from  the  blood  by  passive  filtration.  Heidenhain  believed  it 
to  be  an  exudate  for  the  following  reasons: 

1  It  is  considered  probable  that  hemoglobin  itself  is  the  agent  which  brings  about 
this  decomposition.  Catalase,  which  is  also  present  in  blood,  decomposes  hydrogen 
peroxide  with  the  production  of  inactive,  or  molecular  oxygen. 


ORIGIN  AND  COMPOSITION  OF  LYMPH  363 

If  the  lymph  were  derived  from  the  blood  by  mere  leakage  or 
filtration  through  the  walls  of  the  bloodvessels,  the  rate  of  leakage 
should  be  greater,  the  greater  the  pressure  of  the  blood.  The  rate  of 
flow  of  lymph  in  the  Thoracic  Duct,  however,  does  not  always  decrease 
when  the  arterial  blood-pressure  decreases,  nor  does  it  always  increase 
when  the  arterial  pressure  increases.  Then,  again,  the  injection  of 
strong  salt  solutions  into  the  circulation  might  be  expected  to  with- 
draw fluid  from  the  lymph-spaces  by  osmotic  attraction,  yet  the  lymph- 
flow  from  the  thoracic  duct  is  actually  increased  by  this  procedure. 
Finally  certain  specific  substances,  particularly  crayfish  extract,  and 
extracts  of  leeches  or  shell-fish,  certain  Proteoses  and  also  the  South 
American  arrowhead  poison  Curare  cause  a  very  great  increase  in  the 
flow  of  lymph,  as  Heidenhain  supposed,  by  stimulating  the  secretory 
activity  of  the  vessel-walls  through  which  the  lymph  issues  into  the 
interstices  of  the  tissues. 

Nevertheless  Starling  has  conclusively  demonstrated  that  the  pro- 
duction of  lymph  is,  after  all,  a  process  of  passive  filtration.  The 
phenomena  adduced  by  Heidenhain,  convincing  as  at  first  sight  they 
appear  to  be,  are  nevertheless  simply  attributable  to  .the  fact  that  the 
Permeability  of  the  bloodvessels  for  lymph  varies  very  greatly  in  differ- 
ent parts  of  the  body.  These  differences  in  permeability  lead  to  differ- 
ences in  the  rate  of  filtration  of  lymph  no  less  pronounced  than  the 
difference  in  the  rate  of  filtration  of  water  through  paper  and  through 
unglazed  porcelain.  The  most  permeable  vessels  are  the  capillaries 
in  the  Liver,  while  the  capillaries  in  the  skeletal  muscles  are  almost 
impermeable.  We  can  render  the  capillaries  in  the  leg-muscles  per- 
meable by  heating  them  to  56°  C.,  and  in  this  way  cause  such  extensive 
transudation  of  lymph  that  a  frog's  leg,  so  treated,  becomes  rapidly 
edematous.  If  the  blood-pressure  in  the  liver  be  raised  or  lowered 
the  lymph-flow  is  raised  or  lowered  in  like  proportion,  but  the  pressure 
in  the  liver  and  that  in  the  general  arterial  system  do  not  always  run 
parallel,  so  that  the  departures  from  parallelism  between  arterial  pres- 
sure and  lymph-flow  observed  by  Heidenhain  were  not  inconsistent 
with  the  view  that  lymph  is  a  transudate,  mainly  furnished  by  the 
vessels  of  the  liver.  Strong  salt  or  sugar  solutions  simply  alter  the 
distribution  of  the  interstitial  fluids,  causing  a  general  imbibition  of 
fluid  into  the  vascular  system,  and  a  Hydremic  Plethora  which  results 
in  readjustment  by  more  rapid  filtration  into  the  lymph-spaces  in  the 
liver.  If  we  previously  withdraw  from  the  vascular  system  enough 
blood  to  equal  the  volume  of  fluid  which  is  attracted  into  it  by  the 
subsequent  injection  of  salt  or  sugar,  no  plethora  results,  and  no 
increased  flow  of  lymph  ensues. 

The  various  Lymphagogues  or  lymph-producing  substances  alluded 
to  above  cause  an  increased  transudation  by  the  injury  they  cause  to 
the  walls  of  the  blood-vessels,  greatly  increasing  their  Permeability, 
and  producing  an  effect  analogous  to  that  of  heating  to  56°  C. 

The  composition  of  lymph  is  very  variable.    In  general  it  may  be 


364  VEHICLES  OF  CHEMICAL  CORRELATION 

regarded  as  resembling  blood-plasma,  but  containing  a  larger  propor- 
tion of  tissue  waste-products  and  of  fatty  substances  derived  from  the 
chylous  lymph-vessels  of  the  intestine. 


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THE  COMPOSITION  OF  THE  BLOOD: 

Abderhalden:     Zeit.  f.  physiol.  Chem.,  1897,  23,  p.  521;  1898,  25,  p.  65. 
SERUM-PROTEINS  : 

Reiss:     Beitr.  z,  chcm.  Physiol.  u.  Pathol.,   1904,  4,  p.   150.     Arch.  f.  exp.  Path. 

u.  Pharm.,  1904,  51,  p.  18.     Munch,  med.  Woch.,  1908,  55,  p.  1853.     Jahrb.  f. 

Kinderheilkunde,  1909,  70,  pp.  3,  174.     Ergeb.  d.  inn.  Med.  u.  Kinderheilkunde, 

1913,  10,  p.  531.     Deutsch.  Arch.  f.  klin.  Med.,  1915,  117,  p.  175. 
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Buck:     Jour.  Pharm.  Exp.  Therap.,  1913-14,  5,  p.  553. 
Wells:     Jour.  Biol.  Chem.,  1913,  15,  p.  37. 
Thompson:     Ibid.,  1915,  20,  p.  1. 
Briggs:     Ibid.,  1915,  20,  p.  7. 

Tranter  and  Rowe:     Jour.  Am.  Med.  Assn.,  1915,  65,  p.  1433. 
Rowe:     Jour.  Lab.  Clin.   Med.,    1915-16,  1,  pp.  439  and  485.     Arch.  Int.   Med., 

1916,  17,  p.  455;  1917,  19,  p.  354. 

Righetti:     Univ.  California  Pubs.,  Pathology,  1916,  2,  p.  205. 
Hurwitz  and  Meyer:     Jour.  Exp.  Med.,  1916,  24,  p.  515. 
Schmidt  and  Schmidt:     Jour.  Immunology,  1917,  2,  p.  343. 
Hanson  and  McOuarrie:     Jour.  Pharm.  Exp.  Therap.,  1917-18,  10,  p.  261. 
Hanson:     Jour.  Immunology,  1918,  3,  p.  67. 
Clark:     Ibid.,  1918,  3,  p.  147. 
Toyama:     Jour.  Biol.  Chem  ,  1918-19,  38,  p.  161. 
COAGULATION  OF  THE  BLOOD: 

Wooldridge:     Proc.  Roy.  Soc  ,  London,  1883-4,  36,  p.  417;  1884-50,  38,  pp.  69  and 

260;  1886,  40,  pp.  134  and  320;  1887,  42,  p.  230;  1887-8,  43,  p.  367;  1888,  44,  p. 

282.     Uebersicht  einer  Theorie  der  Blutgerinnung,  Ludwig's  Festschrift,  Leipzig, 

1887,  p.  221.     Arch.  Anat.  u.  Physiol.,  1888,  p.  527.     On  the  Chemistry  of  the 

Blood,  Collected  Papers,  London,   1893. 

Martin:     Jour,  and  Proc.  Roy.  Soc.,  New  South  Wales,  1895. 
Sabbatini:     Arch.  ital.  de  Biol.,  1903,  39,  p.  333. 
Morawitz:     Ergeb.  d.  Physiol.,  1905,  4,  p.  307. 
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p.  10.     Biol.  Bull.,  1902-3,  4,  p.  301.     Hofmeister's  Beitr.,  1904,  5,  pp.  191  and 

534;  1905,  6,  p.  260;  1906,  8,  p.  67;  1907,  9,  p.  185. 
Hekma:     Int.  Zeit.  phys.  chem.  Biol.,  1915,  2,  pp.  279,  299,  352.     Biochem.  Zeit., 

1916,  73,  pp.  370  and  428;  1916,  74,  pp.  63  and  219. 
CHEMISTRY  OF  HEMOGLOBIN: 

Marchlewski:     Die  Chemie  des  Chlorophylls,  1895. 
Gamgee:     Schafer's  Text-book  of  Physiology,  Edinburgh,  1898,  vol.  1. 
Kiister:     Zeit.  f.  Physiol.  Chem.,  1898-9,  26,  p.  314;  1906,  47,  p.  294. 
Nencki  and  Zaleski:     Ber.  d.  d.  chem.  Ges.,  1901,  34,  p.  997. 
Marchlewski:     Zeit.  f.  physiol.  Chem.,  1904,  41,  p.  33. 
Butterfield:     Ibid.,  1912,  79,  p.  439. 

Heubner  and  Rosenberg:     Biochem.  Zeit.,  1912,  38,  p.  345. 
Schumm:     Zeit.  f.  physiol.  Chem.,  1913,  83,  p.  1. 
Newcomer:     Jour.  Biol.  Chem.,  1919,  37,  p.  465. 
CRYSTALLINE  FORM: 

Halliburton:     Quar.  Jour.  Micr.  Sci.,  1887,  28,  pp.   181  and  201. 
Reichert:     Am.  Jour.  Physiol.,  1903,  9,  p.  97. 

Reichert  and  Brown:     Carnegie  Inst.  pubs.  No.  116,  Washington,  1909. 
Loeb  and  Brown:     Science,  N.  S.,  1917,    5,  p.  191. 
Robertson:     The  Physical  Chemistry  of  the  Proteins,  New  York,  1918. 
ORIGIN  AND  COMPOSITION  OF  LYMPH  : 

Starling:     Principles  of  Human  Physiology,  Philadelphia,  1915. 


CHAPTER  XVI. 
EXAMPLES  OF  CHEMICAL  CORRELATION. 

THE   CHEMICAL   CORRELATION    OF   RESPIRATORY   ACTIVITIES. 

The  normal  Respiratory  Movements  of  the  diaphragm  and  inter- 
costal muscles  are  adjusted  to  the  average  need  for  oxygen  which  is 
imposed  by  the  normal  functional  activities  of  our  tissues.  The  per- 
formance of  function  and  the  maintenance  of  the  temperature  of  the 
body  necessitate  an  expenditure  of  energy  which,  since  the  Hydrolyses 
which  occur  in  living  tissues  are  usually  but  slightly  exothermic,  must 
be  derived  for  the  greater  part  from  energy  liberated  by  Oxidations. 
As  the  tissues  which  are  primarily  concerned  in  the  performance  of 
mechanical  work  are  the  muscular  tissues,  variations  of  their  activity 
may  most  clearly  be  seen  to  necessitate  corresponding  variations  in  the 
rapidity  and  extent  of  the  oxidations  upon  which  their  power  of  per- 
forming work  depends.  Of  all  the  various  tissues  of  the  body,  in  fact, 
the  muscles  are  subject  to  the  most  sudden  and  extreme  variations  of 
functional  activity,  being  at  the  one  moment  in  the  state  of  moderate 
tension  which  is  the  normal  condition  of  rest,  and  at  the  next  expending 
all  the  energy  required,  for  example,  to  lift  the  whole  weight  of  the 
body  up  a  steep  incline.  To  provide  a  sufficient  oxygen  supply  to 
render  possible  at  all  times,  without  alterations  of  the  respiratory 
rhythm,  the  maximal  expenditure  of  energy  by  the  skeletal  muscles, 
would  require  a  very  great  wastage  of  energy  by  the  respiratory  muscles 
themselves,  or  else  the  relegation  of  an  excessive  proportion  of  the 
bodily  volume  to  performance  of  respiratory  functions.  The  mechan- 
ism actually  and  normally  employed  provides  an  amplitude  of  oxygen 
for  customary  and  moderate  needs  and  when  the  oxygen  requirements 
of  the  skeletal  muscles  renders  the  customary  means  of  ventilating 
the  body  insufficient,  then  the  efficiency  of  ventilation  is  temporarily 
enhanced  by  a  very  decided  increase  in  the  frequency  and  amplitude 
of  the  respiratory  movements. 

Now  there  is  no  immediate  or  obvious  connection  between  the  move- 
ments of  the  respiratory  muscles  and  those  of  the  skeletal  muscles. 
There  is  no  anatomical  or  mechanical  connection  or  association  be- 
tween them  that  would  render  it  a  priori  probable  that  the  motions  of 
the  one  group  of  muscles  would  tend  to  synchronize  in  frequency  and 
extent  with  those  of  the  other.  Moreover,  the  respiratory  movements 
in  the  adult  higher  vertebrates  are  known  to  be  primarily  under  the 
control  of  a  particular  region  of  the  Medulla  Oblongata,  situated  in 
floor  of  the  fourth  ventricle,  and  designated  the  Respiratory  Center. 


366  EXAMPLES  OF  CHEMICAL  CORRELATION 

Stimulation  of  this  area  enhances  the  rate  and  amplitude  of  the  res- 
piratory movements.  Its  narcotization  or  injury  depresses  or  annuls 
the  respiratory  movements.  The  actual  synchrony  is  therefore  not 
directly  between  the  skeletal  muscles  and  the  respiratory  muscles, 
but  between  the  skeletal  muscles  and  the  nervous  tissues  of  the 
respiratory  center.  Here  we  have  an  even  less  obvious  relationship 
between  tissues  which  nevertheless  act  in  perfection  of  harmony,  and 
the  source  of  this  harmony  lies  in  a  chemical  and  not  in  a  spatial  or 
mechanical  interdependence  of  the  tissues  which  participate  in  it. 

The  initial  effect  of  deprivation  of  oxygen  or  of  interference  by 
mechanical  or  other  means  with  the  entrance  of  air  into  the  lungs  is 
an  increased  amplitude  and  frequency  of  the  respiratory  movements, 
a  condition  which  is  designated  Hyperpnea.  This  is  succeeded  by  the 
stage  of  Dyspnea,  in  which  the  still  more  rapid  movements  become 
almost  convulsive  in  character,  until  finally  every  muscle  which  can 
directly  or  indirectly  assist  in  the  effort  to  fill  or  empty  the  lungs  is 
brought  into  i  itense  activity.  This  activity  is  quite  uncontrollable, 
as  the  reader  may  convince  himself  by  the  simple  endeavor  to  "hold 
the  breath"  for  a  prolonged  period.  If,  finally,  the  lack  of  oxygen,  or 
obstruction  to  the  passage  of  air,  still  defeats  the  object  of  these 
exertions,  a  relatively  sudden  cessation  of  respiratory  convulsions  sets 
in,  due  to  paralysis  of  the  respiratory  center,  and  the  animal  or  man  is 
now  said  to  have  suffered  Asphyxia.  If,  on  the  contrary,  instead  of 
deprivation  of  oxygen  or  obstruction  to  the  intake  or  exit  of  air,  we 
have  an  exceptionally  efficient  ventilation  of  the  lung,  by  forcible  and 
repeated  inflation  or  by  a  series  of  rapid  and  very  deep  voluntary 
breathing  movements,  then  a  condition  of  temporary  suspension  of 
the  activity  of  the  respiratory  center  sets  in,  a  condition  known  as 
Apnea,  which  is  purposely  cultivated  by  divers  and  swimmers  before 
undertaking  a  period  of  prolonged  immersion  below  the  surface  of 
water.  Either  no  desire  to  breathe  is  experienced  for  a  perceptible 
interval,  or  the  desire  is  very  easily  controlled  by  a  voluntary  effort. 

After  a  somewhat  longer  lapse  of  time  than  usual,  however,  the  desire 
to  breathe  is  again  acutely  felt,  and  the  respiratory  movements  there- 
after become  again  uncontrollable  by  any  effort  of  the  will. 

Now  the  effects  of  suspended  breathing  are  twofold.  In  the  first 
place  the  supply  of  oxygen  to  the  blood,  and  therefore  to  the  tissues,  is 
cut  off  and  the  available  oxygen  in  the  body,  free  or  combined  in 
easily  dissociable  compounds  like  Oxyhemoglobin  is  soon  exhausted  by 
the  irreducible  minimum  of  oxidative  change  which  accompanies  the 
life  of  all  the  tissues.  In  the  second  place  the  carbon  dioxide  which 
ultimately  results  from  these  oxidations  cannot  escape  from  the  body, 
and  therefore  accumulates  in  the  blood  and  in  the  tissues. 

The  stimulated  activity  of  the  respiratory  center  which  accompanies 
inadequacy  of  respiration  is  due  to  some  change  in  the  blood  which 
irrigates  it.  This  is  conclusively  shown  by  the  fact  that  if  the  cerebral 
circulations  of  two  animals  be  "crossed,"  so  that  the  blood  from  the 


CHEMICAL  CORRELATION  OF  RESPIRATORY  ACTIVITIES     367 

carotid  artery  of  the  one  animal  supplies  the  brain  of  the  other,  then 
the  prevention  of  effective  respiration  in  the  animal  of  which  the 
brain  is  receiving  normal  blood  produces  no  hyperpnea  or  dyspnea, 
while  the  other  animal,  which  can  breathe  freely,  but  whose  brain  is 
supplied  with  blood  from  an  asphyxiated  animal,  shows  every  sign  of 
respiratory  distress. 

Two  possibilities  evidently  exist,  therefore.  Either  the  stimulation 
of  the  respiratory  center  which  results  from  prevention  of  the  normal 
ventilation  of  the  lung  is  due  to  a  lack  of  sufficient  oxygen  in  the  blood 
which  supplies  the  respiratory  center,  or  else  it  is  attributable  to  the 
accumulation  of  carbon  dioxide  in  the  blood-supply.  It  may  be  that 
both  of  these  factors  play  some  part  in  determining  the  total  result,1 
but  by  far  the  predominant  part  is  that  which  is  played  by  the  accu- 
mulation of  Carbon  Dioxide,  as  will  be  clear  from  the  following  con- 
siderations : 

In  the  first  place  it  has  long  been  known  that  a  very  slight  increase, 
relatively  speaking,  in  the  carbon-dioxide  content  of  the  inspired  air 
leads  to  a  considerable  acceleration  and  increase  in  amplitude  of  the 
respiratory  movements.  To  bring  about  a  like  increase,  by  a  mere 
decrease  of  oxygen,  provided  thorough  ventilation  of  the  lungs  be 
secured  by  unobstructed  breathing  movements,  requires  a  very  much 
greater  diminution  of  oxygen  pressure  than  the  requisite  increase  of 
carbon-dioxide  pressure.  Then,  again,  in  the  performance  of  muscular 
work  there  is  little  or  no  deficiency  of  oxygen  in  the  blood,  but  the 
content  of  carbon  dioxide  must  be  increased,  for  the  output  of  carbon 
dioxide  in  the  lungs  is  increased  and,  furthermore,  the  carbon-dioxjde 
content  of  the  air  contained  in  the  alveoli  of  the  lungs,  the  Alveolar 
Air,  is  increased  by  work.  The  performance  of  work  being  in  fact 
accomplished  by  means  of  the  liberation  of  energy  derived  from  oxi- 
dations, the  end-products  of  these  oxidations,  among  which  carbon 
dioxide  and  water  are  predominant,  must  accumulate  in  the  tissues 
during  the  performance  of  work,  and  therefore  be  more  abundantly 
contained  in  the  blood  than  during  rest.  The  supply  of  oxygen  to  the 
tissues,  on  the  other  hand,  is  normally  superabundant,  and  the  muscular 
tissues,  moreover,  contain  a  certain  reserve-store  of  oxygen,  and  when 
they  are  excised  from  the  body,  they  can  contract  and  perform  work  for 
a  considerable  period  in  an  atmosphere  which  is  devoid  of  oxygen. 
In  fact,  the  saturation  of  the  arterial  Hemoglobin  with  oxygen  is  so 
nearly  complete  in  normal  respiration  that  the  hyperpnea  which 
results  from  energetic  exercise  would  be  devoid  of  utility  if  its  object 
were  the  introduction  of  more  oxygen  to  the  tissues.  Finally,  we 
have  seen  that  Apnea  may  result  from  enhanced  ventilation  of  the 
lungs,  but  this  is  not  due  to  an  increased  intake  of  oxygen  or  of  satur- 
ation of  the  tissues  therewith,  for  it  may  be  brought  about  as  well  by 

1  It  is  improbable  that  lack  of  oxygen  is  in  itself  a  stimulus  to  the  respiratory  center 
or  any  other  tissue.  The  apparent  stimulation,  if  it  occurs  at  all,  is  only  indirectly 
attributable  to  deficiency  of  oxygen. 


368  EXAMPLES  OF  CHEMICAL  CORRELATION 

repeated  and  forced  filling  and  emptying  of  the  lungs  by  an  indifferent 
gas,  such  as  hydrogen  or  nitrogen,  and  when  this  has  been  done,  it  is 
found  that  the  carbon-dioxide  tension,  in  the  alveolar  air  and  therefore 
in  the  arterial  blood,  is  decidedly  lower  than  normal. 

The  increased  carbon-dioxide  tension  of  the  blood  in  obstructed 
breathing  is  therefore  the  stimulus  which  excites  the  activity  of  the 
respiratory  center.  But  the  carbon  dioxide  may  conceivably  act  in 
either  of  two  ways  upon  the  center,  namely  as  a  specific  chemical  stimu- 
lant, or  else  indirectly  by  the  increase  in  the  hydrogen  ion  concentra- 
tion of  the  blood  which  it  brings  about. 

The  experiments  of  Winterstein  were  designed  to  elucidate  this 
question.  This  investigator  introduced  acids  into  the  blood  which  was 
passing  by  the  carotid  artery  to  the  brain,  and  he  obtained  a  decided 
acceleration  of  the  respiratory  rhythm  as  a  result.  Prior  to  these 
experiments  of  Winterstein,  it  had  also  been  shown  that  in  frogs  in 
which  the  floor  of  the  fourth  ventricle  has  been  exposed,  the  direct 
application  of  acids  to  this  area  of  the  medulla  causes  acceleration  of  the 
respiratory  rhythm,  while  that  of  alkalies  slows  it.  Both  experiments 
were  inconclusive,  however,  because  they  did  not  enable  us  to  ascer- 
tain whether  the  acids  administered  excited  the  center  by  virtue  of  the 
hydrogen  ions  which  they  contributed  to  the  blood,  or  by  setting  free 
carbon  dioxide  from  bicarbonates  and  thus  increasing  the  carbon- 
dioxide  tension  of  the  blood.  Subsequent  experiments  by  Laqueur 
and  Verzar  threw  more  light  upon  the  question,  tending  to  show  that 
carbon  dioxide  is  a  specific  stimulant  for  the  respiratory  center,  for  they 
found,  using  Winterstein's  technique,  that  the  nature  of  the  acid 
added  to  the  cerebral  circulation  profoundly  affected  the  result,  and 
the  efficiency  of  the  various  acids  did  not  run  parallel  to  their  "strength" 
or  dissociation  into  ions.  Carbon  dioxide,  lactic  acid  and  various  fatty 
acids  are  much  more  efficient  stimulators  of  respiration  than  the 
strong  mineral  acids.  Evidently,  therefore,  we  have  here  to  deal  with 
an  effect  which  is  not  wholly  a  hydrogen-ion  effect,  but  also  in  part 
an  effect  involving  the  undissociated  molecule  or  the  anions  of  the 
acid  employed* 

THE  CHEMICAL  REGULATION  OF  THE  CIRCULATORY  SYSTEM. 

Removal  of  the  Suprarenal  Glands  in  animals,  or  their  destruction  by 
disease  (usually  tubercular)  in  man,  is  followed  by  the  rapid  appearance 
of  intense  prostration  and  muscular  weakness.  The  blood-pressure 
falls  to  an  extremely  low  level,  and  death  finally  supervenes.  In  man, 
the  destruction  of  the  glands  by  disease  is  usually  somewhat  gradual 
and  the  symptoms  are  correspondingly  slow  to  develop.  They  are  of 
the  same  description  as  those  which  develop  in  animals  when  these 
glands  are  excised,  but,  in  addition,  a  peculiar  patchy  bronze-like  pig- 
mentation of  the  skin  occurs.  The  nature  of  the  pigment  which  is 
deposited  in  these  patches  is  unknown,  but  it  is  highly  probable  that 


CHEMICAL  CORRELATION  OF  RESPIRATORY  ACTIVITIES     369 

it  is  chemically  related  to  Adrenaline,  for  adrenaline,  like  the  Tyrosine 
from  which  it  is  probably  derived,  is  readily  converted  into  highly 
colored  substances  by  oxidizing-agents  and  by  oxidizing-ferments, 
especially  by  the  Tyrosinase  which  occurs  in  many  vegetable  tissues, 
particularly  those  of  fungi,  and  also  in  certain  animal  tissues,  as,  for 
example,  in  tumors  arising  in  the  suprarenal  bodies  (melanomas)  and 
in  the  ink-sac  of  the  cephalopod  sepia. 

The  blood-pressure  raising  substance,  adrenaline,  which  occurs  in 
the  medulla,  or  inner  portion  of  the  suprarenal  gland,  is  capable,  when 
administered  intravenously,  of  correcting  the  excessively  low  blood- 
pressure  in  animals  with  the  suprarenals  excised,  or  in  Addison's  disease, 
but  it  does  not  avail  to  prevent  the  ultimate  death  of  the  animals  and 
it  is  probable  that  other  substances  essential  to  life  are  produced  by 
these  glands  besides  adrenaline.  It  is  possible  that  the  cortex  of  the 
gland,  which  has  an  epithelial  origin  and  differs  both  in  structure  and 
embryological  development  from  the  medulla,  may  play  an  equally 
essential  part  in  the  bodily  economy.  This  is  indicated  in  the  first 
place  by  the  fact  that  serious  symptoms  of  adrenal  insufficiency  may 
accompany  degenerative  changes  affecting  the  cortex  alone,  and  further- 
more by  the  remarkable  effect  of  extensive  superficial  Burns  upon  the 
cortex.  Burns  or  scalds,  if  at  all  extensive,  are  followed  by  lesions  of 
the  suprarenal  cortex  and  especially  by  minute  hemorrhages  therein. 
These  changes  are  progressive  for  several  days  following  the  injury,  and 
are  prominent  in  instances  of  deferred  death  resulting  from  extensive 
burns  or  scalds.  The  unusual  abundance  of  Lipoids  and  especially  of 
Cholesterol  Esters  in  the  suprarenal  cortex  is  suggestive  of  a  function 
related  to  the  lipoid  metabolism,  but  the  nature  of  this  function 
remains  unknown. 

It  is  of  the  active  physiological  principle  of  the  medulla,  namely 
Adrenaline,  that  our  knowledge  is  most  extensive.  This  substance, 
when  injected  intravenously  in  minute  amounts  (0.001  mg.  and  upward 
in  a  dog),  causes  a  marked  rise  in  blood-pressure  (Fig.  22).  This 
phenomenon  is  one  of  many  consequences  of  the  general  action  of 
adrenaline  in  stimulating  the  Myoneural  Junctions  of  the  muscles  inner- 
vated by  the  sympathetic  system.  The  action  is  not  upon  the  nerves 
themselves,  or  upon  their  anatomically  visible  endings,  for  it  is  more 
and  not  less  pronounced  when  the  nerve  is  cut  and  allowed  to  degener- 
ate up  to  and  including  its  anatomical  connection  with  the  muscle. 
The  action  is  not  upon  muscle-fibers  themselves,  for  in  the  first  place 
muscles  not  innervated  from  the  sympathetic  system  are  not  affected 
by  adrenaline,  and  in  the  second  place  the  muscles  innervated  by  the 
sympathetic  system  are  not  all  affected  alike,  for  if  inhibitory  fibers 
predominate  the  muscle  is  relaxed,  while  if  stimulatory  fibers  predomi- 
nate the  muscle  is  contracted.  The  glandular  tissues  are  variously 
affected,  for  if  the  adrenaline  stimulates  their  secretory  activity,  the 
contraction  of  the  bloodvessels  and  the  consequently  diminished  blood- 
supply  operate  in  a  contrary  direction.  In  the  kidneys  the  diminished 
24 


370  EXAMPLES  OF  CHEMICAL  CORRELATION 

blood-supply  at  first  reduces  the  output  of  urine,  but  when  the  blood- 
pressure  effect  passes  off,  which  it  does  rather  rapidly,  a  decided 
Diuresis  follows. 

Local  subcutaneous  administration  of  adrenaline  so  constricts  the 
adjacent  vessels  that  its  absorption  is  thereby  much  delayed  and  its 
action  is  prolonged.  It  is  upon  this  fact  that  the  extensive  employ- 
ment of  adrenaline  in  minor  surgical  operations  depends.  Bleeding  is 
prevented,  and  an  unobstructed  view  of  the  tissues  is  secured  for  the 
period  of  the  operation.  Furthermore,  local  Anesthetics  simultaneously 
applied  share  in  the  difficulty  of  absorption,  and  therefore  continue 
their  local  analgesic  action  for  a  longer  period  than  would  otherwise  be 
attainable.  The  tendency  to  post-operative  hemorrhage  is,  however, 
said  to  be  enhanced  by  adrenaline  and  it  is  also  to  be  remembered  that 


FIG.  22. — Blood-pressure  (B.P.}  and  bowel  volume  (7.7.)  of  cat.  At  A  injection  of 
adrenaline.  The  blood-pressure  rises  and  bowel  volume  diminishes,  indicating  con- 
striction of  the  mesenteric  vessels.  As  these  relax  again  the  blood-pressure  falls.  The 
vagi  had  been  divided  previously,  so  that  there  is  no  secondary  slowing  of  the  heart. 
(After  Cushny.) 

the  normal  defense  of  the  tissues  against  infections  is  supplied  by  the 
blood  and  by  the  leukocytes  which  the  blood  and  lymph  contain,  so 
that  a  measure  of  natural  protection  against  bacterial  invasions  is 
denied  the  tissues  by  this  procedure. 

An  important  affect  of  intravenous  injections  of  adrenaline  is  the 
appearance  of  Glucohemia  and  its  resultant,  Glycosuria.  The  power  of 
the  liver  to  polymerize  glucose  is  apparently  rendered  deficient  and  the 
normal  equilibrium  between  glycogen  and  glucose  in  the  liver-tissues  is 
shifted  in  favor  of  the  glucose. 

It  has  been  established  in  many  ways  that  minute  quantities  of 
adrenaline  are  constantly  present  in  the  blood.  That  this  must  be 
continually  supplied  to  the  blood  by  the  suprarenal  glands  follows  from 
the  fact  that  injected  adrenaline  very  rapidly  disappears  from  the 
circulation  and  the  tissues,  being  apparently  destroyed  or,  at  all  events, 


CHEMICAL  CORRELATION  OF  PROCESSES  OF  DIGESTION    371 

converted  into  substances  devoid  of  the  typical  activities  of  adrenaline. 
It  is,  however,  a  question  that  is  still  being  debated  whether  or  not  the 
small  amounts  normally  present  in  the  blood-stream  actually  influence 
the  tone  of  the  vascular  system,  and  help  to  maintain  the  normal 
blood-pressure.  The  extremely  low  blood-pressure  in  Addison's 
disease,  however,  and  the  marked  effect  of  adrenaline  in  raising  it, 
stated  even  to  be  more  marked  than  in  normal  individuals,  would 
seem  to  point  rather  decisively  to  a  constant  relationship  between  the 
functional  activity  of  the  suprarenals  and  the  maintenance  of  normal 
blood-pressure. 

According  to  Cannon,  however,  one  of  the  most  important  functions 
of  the  suprarenals  is  to  assemble  a  group  of  conditions  appropriate  for 
the  defense  of  the  organism  in  an  emergency.  Violent  Emotional 
States,  such  as  fear,  rage  or  pain  (and  also  anesthesia)  lead  to  a  marked 
discharge  of  adrenaline  from  the  suprarenal  glands,  and  to  all  the  effects 
which  arise  from  intravenous  injection  of  adrenaline.  The  intravenous 
injection  of  adrenaline  in  the  cat  will,  as  a  matter  of  fact,  elicit  very 
many  of  the  most  easily  recognizable  external  signs  of  fear  without  the 
application  of  any  other  stimulus.  Thus  the  hair  of  the  back  and  tail 
is  raised,  and  the  pupils  of  the  eyes  are  widely  dilated.  It  is  to  the 
presence  of  an  excess  of  adrenaline  in  the  blood  that  the  glycosuria  of 
violent  emotions,  the  so-called  Emotional  Glycosuria,  is  partly  due.  It 
has  been  shown  by  Macleod  that  stimulation  of  the  splanchnic  nerves, 
which  innervate  the  suprarenal  glands,  results  in  the  production  of 
glucohemia  which  is  partially  attributable,  however,  to  the  direct  stimu- 
lation of  the  liver  through  the  hepatic  branches  of  the  splanchnics. 

The  effect  of  emotional  stimulation,  operating  through  the  splanch- 
nics, is  to  increase  the  adrenaline  in  the  blood  and  thereby  to  increase 
the  blood-pressure,  quicken  the  heart-beat  and  thus  enhance  the  mobil- 
ity of  the  blood  and  the  rate  of  access  and  exit  of  the  raw  materials 
and  products  of  metabolic  activities.  The  liability  to  external  hemor- 
rhages is  reduced  owing  to  the  constriction  of  peripheral  vessels,  and 
also  to  a  definite  reduction  of  the  coagulation-time  of  the  blood,  which 
is  another  result  of  adrenaline  administration.  The  instantly  avail- 
able nutritive  materials  for  the  muscle-cells  are  increased  by  the 
mobilization  of  sugar-reserves.  In  short  the  animal  is  placed  in  the 
best  attainable  condition  for  a  sudden  extreme  effort  and  the  sustain- 
ment  of  possible  injury.  In  conflicts,  or  in  efforts  to  escape  from  more 
powerful  predatory  forms,  the  suprarenal  glands  probably  constitute 
an  essential  factor  in  success  or  failure. 

THE  CHEMICAL  CORRELATION  OF  THE  PROCESSES  OF 
DIGESTION. 

The  arrival  of  foodstuffs  in  the  stomach  is  preceded  by  a  considerable 
secretion  of  Gastric  Juice,  and,  in  consequence,  the  processes  of  gastric 
digestion  are  enabled  to  go  forward  without  delay.  The  correlation 


372  EXAMPLES  OF  CHEMICAL  CORRELATION 

between  the  acts  involved  in  the  intake  of  foodstuffs  and  the  secretory 
activity  of  the  glands  of  the  gastric  mucosa  is,  however,  as  the  classical 
researches  of  Pawlow  have  shown,  nervous  in  origin,  and  not  chemical, 
arising  in  part  from  reflexes  arising  from  optical  and  olfactory  stimuli 
and  in  part  from  gustatory  and  tactile  impressions.  The  detailed 
consideration  of  their  mechanism  belongs  therefore  to  the  domain  of 
physiology  rather  than  to  that  of  biochemistry.  The  subsequent  steps 
in  the  process  of  digestion  involve,  however,  a  very  remarkable  series 
of  chemical  correlations. 

During  gastric  digestion  the  pyloric  sphincter  remains  closed,  and  it 
opens  to  permit  the  discharge  of  the  stomach  contents  only  when  the 
digestion  of  the  proteins  has  attained  the  stage  of  nearly  complete 
conversion  into  Proteoses  or  Peptones.  The  mechanism  which  regulates 
the  tone  of  the  sphincter  is  nervous,  but  the  stimulus  which  releases 
the  reflex  dilatation  is  chemical,  and  consists  of  the  presence  in  the 
lower  end  of  the  stomach  of  foodstuffs  containing  a  definite  excess  of 
hydrogen  ions.  This  is  very  clearly  shown  by  the  investigations  of 
Cannon,  who  found  that  the  period  which  elapses  before  the  first  open- 
ing of  the  sphincter  and  discharge  of  Chyme  into  the  intestine,  is  pro- 
portional to  the  quantity  of  substances  in  the  food  which  are  capable 
of  neutralizing  acids.  Thus,  solutions  of  sugar  or  starches  are  retained 
for  but  a  brief  time  in  the  stomach,  but  the  period  of  their  retention 
may  be  enhanced  very  greatly  by  admixture  with  substances  which 
neutralize  free  acids.  Meat  and  other  dietary  constituents  which 
contain  proteins  on  the  contrary  are  retained  for  a  relatively  prolonged 
period. 

When  the  acid  chyme  has  been  discharged  into  the  duodenum  in 
sufficient  quantity  to  induce  a  certain  acidity  of  the  contents  of  the 
upper  part  of  the  small  intestine,  the  pyloric  sphincter  again  closes  in 
accordance  with  the  general  law  governing  the  musculature  of  the 
intestine,  namely,  that  any  localized  stimulus  causes  relaxation  below 
and  contraction  above  the  stimulated  point. 

When  the  Chyme  is  being  discharged  from  the  stomach  through  the 
dilated  pyloric  sphincter,  an  augmented  outflow  of  Pancreatic  Juice 
is  already  travelling  down  the  pancreatic  duct  to  meet  it.  The  time- 
relations  of  the  production  of  the  two  digestive  fluids,  gastric  juice  and 
pancreatic  juice,  is  illustrated  by  the  following  data  obtained  by 
Pawlow. 

Time  after                                                               Gastric  secretion  Pancreatic  secretion 
partaking  of  food.                                               after  100  grams  of  meat.          after  600  c.c.  of  milk. 

1  hour 11.2  8.8 

2 ' 8.2  7.5 

3 ......       4.0  22.5 

4 \    - ...      1 . 9  9.0 

5  ...........       0.1  2.0 

the  maximal  secretion  of  pancreatic  juice  coinciding,  in  time  with  the 
moment  when  the  maximal  quantity  of  chyme  is  leaving  the  stomach. 


CHEMICAL  CORRELATION  OF  PROCESSES  OF  DIGESTION    373 

The  immediate  origin  of  this  phenomenon  resides  in  the  acidity  of 
the  gastric  contents  which,  upon  the  opening  of  the  pylorus,  come  in 
contact  with  the  mucosa  of  the  upper  part  of  the  duodenum,  and,  in 
fact,  a  copious  secretion  of  pancreatic  juice  may  be  elicited  by  simply 
bathing  the  duodenum  with  dilute  acids,  for  example  0.4  per  cent, 
hydrochloric  acid.  The  same  result  is  obtained  if  the  acid  be  intro- 
duced into  the  jejunum,  but  not  when  it  is  introduced  into  the  ileum. 
The  exciting  agent,  however,  is  not  the  acid  itself,  for  the  injection  of 
0.4  per  cent,  hydrochloric  acid  (one-tenth  normal)  into  the  circulation 
is  without  effect  upon  the  secretion  of  pancreatic  juice.  The  excitation 
of  the  pancreas  is,  on  the  other  hand,  not  accomplished  through  a 
nervous  reflex  because  it  occurs,  and  is  undiminished  when  the  portion 
of  the  intestine  which  is  treated  with  acid  is  isolated  from  all  nervous 
connections,  and  furthermore,  it  continues  after  the  administration  of 
Atropine,  which  paralyzes  the  endings  of  the  secretomotor  nerves. 

The  actual  intermediary  which  brings  about  this  correlation  is  a 
substance  Prosecretin  which  is  present  in  the  mucous  membrane  of  the 
duodenum  and  the  jejunum,  and  which  is  changed  by  acids  into 
Secretin,  a  diffusible,  water-soluble,  heat-resistant  substance,  which  has 
the  property  of  specifically  stimulating  the  secretory  cells  of  the 
pancreas.  If  the  mucous  membrane  be  scraped  from  the  surface  of  the 
duodenum  and  rubbed  up  in  physiological  saline  solution  (0.9  per  cent. 
NaCl)  the  filtered  extract  which  is  thus  obtained  may  be  injected  into 
the  circulation  without  eliciting  any  secretion  of  pancreatic  juice.  If, 
however,  the  extract  be  previously  boiled,  or  acidified  and  then 
neutralized,  the  injection  will  now  be  followed  by  a  copious  secretion 
of  pancreatic  juice.  In  normal  digestion  the  transformation  of  the 
prosecretin  in  the  duodenal  mucosa  into  secretin  is  accomplished  by 
the  acid  chyme,  and  the  secretin  which  is  formed  is  carried  by  the  blood- 
stream to  the  cells  of  the  pancreas. 

Secretin  occurs  in  the  mucosa  of  the  intestine  in  all  vertebrates  and 
even  in  the  intestines  of  fishes.  It  is  diffusible,  is  not  destroyed  by 
boiling,  and  is  soluble  in  acidified  solutions  of  mercuric  chloride,  being 
precipitated  on  neutralization.  It  appears  to  be  a  nitrogenous  base, 
and  is  probably  an  amine  derived  by  Decarboxylization  from  an  amino- 
acid  or  from  an  amino-acid  derivative.  Acidified  extracts  of  the 
intestinal  mucosa  and  of  many  other  tissues,  contain  /3-Iminazolyl 
Ethylamine  but  this  substance  is  devoid  of  action  upon  the  secreting 
cells  of  the  pancreas.  The  chemical  identity  of  secretin  has  therefore 
not  been  established.  A  nitrogenous  base  having  a  similar  action  upon 
the  pancreas  is  known,  however,  namely  Pilocarpine,  a  trimethyl 
ammonium  derivative  obtained  from  the  leaves  of  Pilocarpus  jaborandi. 

It  must  be  stated,  however,  that  acids  are  not  the  only  substances 
which  will  bring  about  a  secretion  of  pancreatic  juice  when  they  come 
into  contact  with  the  duodenal  mucosa.  Fats  are  particularly  active 
in  causing  secretion  of  the  pancreatic  juice  after  their  entry  into  the 
duodenum,  probably,  however,  only  after  they  have  been  partially 


374  EXAMPLES  OF  CHEMICAL  CORRELATION 

converted  into  soaps.  The  origin  of  this  effect  is  unknown.  The 
Soaps,  like  other  Calcium  Precipitants  are  strong  stimulators  of  nerve 
fibers  and  nerve  endings,  and  the  contention  of  Pawlow,  that  their 
action  upon  pancreatic  secretion  arises  reflexly  through  stimulation  of 
nerve  endings  in  the  intestine,  is  therefore  not  unfounded.  On  the 
other  hand  it  has  been  suggested  that  the  soaps  formed  from  fats  in  the 
intestine,  convert  prosecretin  into  secretin  or  into  some  substance  of 
like  action,  which  is  carried  to  the  pancreas  by  the  blood-stream. 
Other  substances  causing  an  especially  abundant  flow  of  pancreatic 
juice  are  Chloral  Hydrate  and  Ethyl  Alcohol. 

The  chemical  coordination  of  the  processes  of  digestion  does  not 
end,  however,  with  the  coordination  of  the  secretory  activities  of 
the  digestive  glands.  If  care  be  taken  to  excise  the  pancreas  without 
allowing  the  tissues  to  come  into  contact  with  the  mucous  membranes 
of  the  intestine,  or  if  the  secretin  is  collected  by  means  of  a  cannula 
placed  in  the  duct,  so  that  it  is  obtained  before  it  touches  the  intestinal 
surface,  it  is  found  that  the  fluid  is  devoid  of  proteolytic  activity. 
Yet  the  moment  after  it  arrives  within  the  intestine  a  very  intense 
proteolytic  activity  is  developed.  The  reason  for  this  is  that  Trypsin 
is  not  present  within  the  tissues  or  secretions  of  the  pancreas  as  such, 
but  in  the  form  of  a  proteolytically  inactive  precursor  which  is  desig- 
nated Trypsinogen.  The  conversion  of  trypsinogen  into  trypsin  will 
not  occur  spontaneously,  under  aseptic  conditions,  even  after  a  period 
of  weeks  or  months.  If,  however,  the  fluid  is  momentarily  acidified 
and  then  neutralized,  the  conversion  of  trypsinogen  into  trypsin  is 
found  to  have  been  completed  within  the  brief  period  of  exposure  to 
the  action  of  hydrogen  ions.  A  more  prolonged  exposure  results  in 
partial  or  complete  destruction  of  the  trypsin,  and  since  the  rate  of 
secondary  destruction  of  the  enzyme  is  proportional  to  the  dissociation 
or  "  strength' '  of  the  free  acid,  it  is  safer  to  employ,  for  the  conversion 
of  the  trypsinogen,  a  weakly  dissociated  acid,  such  as  Salicylic  Acid, 
which  furnishes  a  sufficiency  of  hydrogen  ions  to  activate  the  trypsino- 
gen but  decomposes  the  active  trypsin  relatively  slowly. 

In  actual  digestion  the  activation  of  the  trypsinogen  may  be  brought 
about  in  part,  it  is  true,  by  the  admixture  of  the  pancreatic  juice  with 
the  acid  chyme,  for  the  contents  of  the  duodenum  are  acid  to  Litmus, 
although  alkaline  to  Methyl  Orange,  throughout  the  greater  part  of 
its  length.  But  that  another  chemical  mechanism  exists  in  the  intes- 
tine which  is  capable  of  bringing  about  very  rapid  and  complete  activa- 
tion of  trypsinogen  is  shown  by  the  fact  that  when  the  pancreatic 
secretion  is  poured  into  the  empty  intestine,  the  trypsinogen  which  it 
contains  is  found  to  have  been  activated  within  a  very  brief  period 
after  its  arrival  within  the  intestine;  in  fact  mere  contact  with  the 
surface  of  the  intestinal  mucosa  for  a  few  moments  suffices  to  bring 
about  a  considerable  degree  of  activation,  and  under  such  circumstances, 
of  course,  the  reaction  of  the  fluid  remains  consistently  alkaline. 

This  activation  is  brought  about  by  a  substance  which  is  contained 


CHEMICAL  CORRELATION  OF  PROCESSES  OF  DIGESTION     375 

in  the  secretions  of  the  intestinal  glands  comprising  tlje  so-called  Succus 
Entericus.  The  activating  constituent  is  designated  Enterokinase, 
and  because  of  the  fact  that  it  is  destroyed  or  inactivated  by  heat- 
ing, and  is  furthermore  active  in  very  small  quantities,  it  has  been 
generally  assumed  to  be  an  enzyme,  and  in  fact  Pawlow  has  termed  it  a 
"ferment  of  ferments."  Nevertheless  the  proof  that  enterokinase  is 
an  enzyme  is  very  imperfect.  Many  substances  are  modified  by  heat 
which  are  not  enzymes,  of  course,  and  the  small  amount  of  the  material 
required  to  activate  a  large  volume  of  pancreatic  juice  may  merely  be 
expressive  of  the  minute  quantity  of  trypsin  which  is  actually  present 
in  the  secretion  of  the  pancreas.  We  have  no  method  of  quantitatively 
estimating  trypsinogen  and  enterokinase  except  in  terms  of  each  other 
and  we  have  no  data  which  could  enable  us  to  arrive  at  an  estimate  of 
the  actual  weight  of  trypsinogen  which  is  activated  by  a  given  weight 
of  enterokinase.  More  conclusive  evidence  of  the  enzymatic  character 
of  enterokinase  would  be  afforded  if  we  were  to  find  that  a  limited 
quantity  of  succ  is  e  itericus  will  activate  very  large  quantities  of 
pancreatic  juice  provided,  only,  that  sufficient  lapse  of  time  be  allowed 
for  the  completion  of  the  process.  But  from  the  results  of  Hamburger, 
Hekma  and  others,  it  appears  that  the  contrary  is  actually  the  case, 
and  that  there  is  a  quantitative  relationship  between  the  amount  of 
succus  entericus  which  is  added  to  pancreatic  juice  and  the  amount 
of  trypsin  which  is  produced. 

We  have  stated  that  the  pancreatic  juice,  as  produced  by  the  secre- 
tory cells  of  the  pancreas,  is  proteolytically  inactive.  While  this  is 
generally  the  case,  it  is*not  necessarily  or  invariably  so.  The  juice 
obtained  by  the  action  of  Secretin  is,  it  is  true,  invariably  inactive,  but 
juice  obtained  by  stimulation  of  the  secretomotor  fibers  in  the  vagus 
usually  contains  active  trypsin,  and  juice  containing  preactivated 
trypsin  may  also  be  obtained  after  the  administration  of  certain  food- 
stuffs, particularly  diets  containing  a  high  proportion  of  meat.  The 
seat  and  mechanism  of  this  activation  is  unknown. 

In  general,  however,  it  is  evident  that  the  proteolytic  powers  of 
pancreatic  juice  must  be  much  enhanced  by  admixture  with. succus 
entericus,  in  part  through  the  activation  of  trypsinogen  by  entero- 
kinase and  in  part  owing  to  the  fact  that  succus  entericus  contains 
Erepsin,  an  enzyme  capable  of  splitting  peptones  or  casein,  but  not 
other  proteins,  to  amino-acids.  The  digestion  of  protein  by  mixed 
proteolytic  enzymes  is  always  more  rapid  and  complete  than  when  a 
single  enzyme  is  present,  because  different  enzymes  attack  different 
linkages  preferentially  so  that  in  the  presence  of  two  or  more  enzymes 
a  larger  number  of  amino-acid  linkages  are  rendered  susceptible  to  rapid 
disruption.  This  being  the  case  it  is  a  fact  of  interest  and  importance 
that  pancreatic  juice  itself,  according  to  Pawlow,  stimulates,  by  its 
presence,  the  secretion  of  succus  entericus.  At  all  events  during  gastric 
digestion  the  secretion  of  fluid  from  the  glands  of  the  intestine  is  very 
small,  but  after  passage  of  the  chyme  into  the  intestine  and  the  coinci- 


376  EXAMPLES  OF  CHEMICAL  CORRELATION 

dent  inflow  of  pancreatic  juice  and  bile,  the  secretion  of  succus  entericus 
is  greatly  increased.  According  to  some  observers,  however,  this 
increase  is  attributable  to  secretin,  which  is  believed  by  them  to 
stimulate  the  intestinal  glands  as  it  does  the  glandular  cells  of  the 
pancreas.  It  is  difficult  at  present  to  disentangle  these  alternative 
possibilities,  and  further  investigation  is  evidently  required  before  the 
relative  parts  played  by  secretin  and  the  pancreatic  juice  itself  in 
promoting  the  secretion  of  succus  entericus  can  be  correctly  evaluated. 


THE  CHEMICAL  CORRELATION  OF  THE  ORGANS  OF 
GENERATION. 

The  secondary  sexual  characters  of  the  male,  such  as  the  growth  of 
the  beard  and  the  deepening  of  the  voice  in  man,  the  development  of 
horns  ia  the  ram  and  of  the  comb  and  tail-feathers  of  the  cock,  have 
long  been  known  to  be  attributable  to  the  development  of  the  Testes. 
Castration  has  long  been  practised  both  in  man  and  in  animals  for  the 
purpose  of  preventing  the  development  of  secondary  sexual  characters, 
and  of  bringing  about  the  psychic  and  metabolic  modifications  which 
also  accompany  the  excision  of  these  organs.  The  removal  of  the  testes 
in  man  before  the  onset  of  puberty  prevents  the  appearance  of  the 
beard  and  the  deepening  of  the  voice  which  characterises  that  period 
of  development,  and  hardening  of  the  epiphyses  of  the  bones  is  delayed, 
so  that  the  legs  and  arms  grow  to  an  unusual  length  in  proportion  to 
the  size  of  the  whole  body.  In  certain  varieties  of  sheep  only  the  males 
are  possessed  of  horns,  and  in  these  varieties  castration  of  the  young 
male  altogether  suppresses  the  development  of  the  horns.  Similarly 
the  castration  of  cocks  suppresses  the  development  of  the  comb.  If, 
however,  the  excised  testicle  be  implanted  in  another  part  of  the  body, 
as,  for  example,  in  the  peritoneal  cavity,  then  the  secondary  sexual 
characters  develop  normally,  the  penis  grows  to  its  normal  dimensions, 
the  seminal  vesicles  and  the  prostate  develop  as  if  the  testes  were 
actually  functioning  as  a  generative  organ,  and  yet,  not  only  are  the 
testes  prevented  by  lack  of  communication  with  the  Vas  Deferens  from 
discharging  spermatozoa  but,  as  a  matter  of  fact,  the  spermatogenic 
tissues  of  the  testes  dwindle  away,  and  the  production  of  spermatozoa 
actually  ceases.  The  effect  of  this  organ  upon  the  development  of  the 
secondary  sexual  characters  is  therefore,  evidently,  not  attributable 
to  its  spermatogenic  tissues,  and  appears  to  be  due  to  the  Interstitial 
Cells  which  are  normally  present  between  the  seminal  tubules  and 
become  increased  in  number  in  the  transplanted  organ.  Since  these 
tissues  are  provided  with  no  duct  for  the  conduction  of  their  products 
to  the  exterior,  the  channel  of  transmission  of  the  substances  from  the 
interstitial  cells,  or,  as  Steinach  calls  them  collectively  "  The  puberty- 
gland,"  to  the  tissues  which  they  affect,  can  only  be  the  general  circu- 
lating media,  the  blood  and  lymph.  The  puberty-gland  is,  in  fact,  an 
example  of  the  Ductless  Glands,  or  Endocrine  Organs. 


CHEMICAL  CORRELATION  OF  ORGANS  OF  GENERATION     377 

In  the  female,  the  excision  of  the  ovaries  leads  to  a  more  or  less 
pronounced  tendency  toward  the  acquirement  of  masculine  character- 
istics. Very  marked  effects  upon  the  male,  however,  are  elicited  if 
the  ovary  be  transplanted  into  the  tissues  of  a  castrated  animal  of  the 
same  species.  In  this  case  not  only  do  the  secondary  sexual  characters 
of  the  male  fail  to  develop,  but  those  of  the  female  take  their  place, 
even  to  the  development  of  the  Mammary  Glands.  Here,  again,  the 
effect  appears  to  be  attributable  rather  to  the  interstitial  elements  of 
the  ovary,  than  to  the  reproductive  elements. 

A  remarkable  instance  of  the  converse  effect,  namely,  suppression  of 
female  characteristics  by  secretions  from  the  male  organs  of  generation, 
is  supplied  by  the  sterility  which  is  almost  the  invariable  rule  in  the 
females  of  heterosexual  twins  in  cattle.  A  female  of  this  type  is  known 
to  cattle-breeders  as  a  Free-Martin.  It  has  been  ascertained  by  F.  R. 
Lillie  that  in  cattle  a  twin  pregnancy  is  almost  always  a  result  of  the 
fertilization  of  an  ovum  from  each  ovary  and  development  begins 
separately  in  each  horn  of  the  uterus.  The  ova,  in  the  course  of  devel- 
opment, however,  meet  and  fuse,  and  the  bloodvessels  from  each  side 
anastomose  in  the  connecting  part  of  the  chorion,  so  that  each  embryo 
receives  part  of  its  blood-supply  from  the  other.  Both  the  arterial  and 
venous  circulations  overlap,  so  that  a  constant  interchange  of  blood 
takes  place.  If  both  are  males  or  both  are  females  no  harm  results; 
but  if  one  is  a  male  and  the  other  female,  the  reproductive  system  of  the 
female  is  largely  suppressed  in  its  development,  and  certain  male 
organs  even  develop  in  the  female.  The  effect  of  this  is  to  render  the 
female  incapable  of  reproduction. 

A  recurrent  cycle  of  changes  occurs  in  the  Ovary  of  the  adult  female 
which  results  in  the  intermittent  discharge  of  mature  egg-cells  from  the 
ovarian  tissues  into  the  Fallopian  tubes  leading  into  the  cavity  of  the 
uterus.  The  ovarian  tissues  contain  a  number  of  vesicles,  lined  with 
epithelium  and  each  containing  an  ovum,  which  migrate  toward  the 
surface  of  the  ovary,  at  the  same  time  increasing  in  size.  These  are 
the  Graafian  Follicles,  which  periodically  rupture,  discharging  the  ova 
which  they  contain.  The  discharge  of  the  egg  into  the  Fallopian  tubes 
may  or  may  not  coincide  with  the  period  of  menstruation,  in  fact  such 
evidence  as  we  possess  tends  to  show  that  the  two  processes,  while 
coinciding  approximately  in  frequency,  do  not  occur  with  strict  syn- 
chrony. The  ruptured  Graafian  follicle,  after  the  discharge  of  the 
ovum,  undergoes  a  series  of  degenerative  changes  which  culminate  in 
the  formation  of  the  Corpora  Lutea,  which  when  mature  appear  as 
spherical  masses  of  yellowish  cells,  disposed  in  a  more  or  less  columnar 
manner,  the  columns  of  cells  radiating  from  the  center. 

The  Menstrual  Fluid  in  ma»  consists  of  blood  and  shreds  of  cast-off 
uterine  epithelium,  diluted  by  the  secretions  of  the  mucous  glands  of 
the  uterus.  It  contains  a  very  high  percentage  of  Calcium  and  for  this 
reason  Blair  Bell  has  suggested  that  it  may  be  related  phylogenetically 
to  the  egg-shell  of  birds  or  of  a  remote  common  ancestor  of  the  birds 


378  EXAMPLES  OF  CHEMICAL  CORRELATION 

and  mammals.  However  this  may  be,  a  considerable  storage  of  cal- 
cium occurs  in  the  tissues  of  the  female  prior  to  menstruation,  and  this 
excess  of  calcium  is  suddenly  discharged  during  the  period  of  menstrua- 
tion. Having  regard  to  the  immense  importance  of  the  precise  value 
of  the  §^  ratio  in  determining  the  susceptibility  of  nervous  tissues  to 
stimuli,  it  appears  not  unlikely  that  some  of  the  nervous  accompani- 
ments of  menstruation,  and  particulirly  the  hyperirritability  of  the 
uterus  which  leads  to  the  phenomenon  of  painful  menstruation  or 
Dysmenorrhea  may  be  attributable  in  part  to  the  sudden  reduction  of 
the  calcium-content  of  the  tissues  which  occurs  at  this  period. 

The  menstrual  blood  usually  does  not  clot  at  all,  or  if  it  clots  it  does 
so  very  slowly.  This  remains  the  case  even  if  fibrinogen  be  added  to 
it,  and,  as  .we  have  seen,  calcium  is  not  lacking.  It  can  hardly  be 
deficient  in  kephalin,  or  thrombokinase,  since  the  fluid  contains  so 
much  material  arising  from  the  breaking  down  of  the  tissues  lining  the 
cavity  of  the  uterus.  It  appears  likely,  therefore,  that  the  mucous 
secretions  of  the  uterus  contain  a  substance  similar  to  Antithrombin  or 
hirudin  in  its  action  upon  the  coagulation  of  blood. 

When  the  fertilized  egg  becomes  imbedded  in  the  wall  of  the  uterus  a 
proliferation  of  the  uterine  wall  results  in  the  outgrowth  of  a  Placenta 
which  subsequently  provides  the  developing  embryo  with  circulating 
blood  derived  from  the  mother.  We  have  here  a  remarkably  exact 
coincidence  of  events  and  we  are  led  to  inquire  why  the  tissues  of  the 
uterus  are  aroused  to  the  production  of  this  outgrowth  at  the  very 
moment  when  it  is  about  to  be  required? 

An  answer  to  this  question  has  been  afforded  by  the  very  important 
discoveries  of  L.  Loeb.  This  observer  has  found  that  in  the  female 
guinea-pig,  for  a  period  of  some  ten  days  following  the  phenomenon  of 
Ovulation,  any  injury  to  the  uterine  wall  results  in  the  outgrowth  of  a 
placenta.  The  injury  may  be  of  the  nature  of  a  slight  incision,  in 
which  case  a  localized  growth  occurs  which  may  be  duplicated  at  other 
points  in  the  uterus,  so  that  as  many  as  twenty  different  placentae  may 
be  formed  in  this  way  in  a  single  uterus.  Or  the  injury  may  consist  of 
the  irritation  afforded  by  the  presence  of  a  foreign  object,  such  as  a 
thin  glass  rod  or  a  number  of  particles  of  paraffin.  In  this  case  the 
growth  of  placental  tissue  may  become  so  great  as  to  interfere  with  the 
nutrition  of  the  newly  formed  tissue  and  induce  its  degeneration  and 
autolysis.  The  formation  of  placentae  is  prevented  if  the  ovaries  are 
extirpated  or  even  if  the  Corpora  Lutea  which  they  contain  are  excised. 
The  stimulus  which  arouses  this  reaction  of  the  uterus  to  mechanical 
irritation  comes,  therefore,  from  the  corpora  lutea.  If  the  corpora  lutea 
are  not  excised  at  once,  and  placentse  are  permitted  to  form,  they  attain 
a  smaller  size  and  degenerate  more  rapidly  if  the  ovaries  or  the  corpora 
lutea  are  excised  before  their  full  development  is  attained. 

Among  the  many  correlations  which  underlie  and  render  possible 
the  development  of  the  embryo,  the  next  into  which  we  have  attained 
some  measure  of  insight  is  that  which  obtains  between  the  development 


CHEMICAL  CORRELATION  OF  ORGANS  OF  GENERATION    379 

of  the  embryo  and  the  development  of  the  Mammary  Glands  of  the 
mother.  .As  the  fetus  grows  the  mammary  glands  of  the  pregnant 
female  hypertrophy  until  a  portion  of  the  hypertrophic  tissue  begins  to 
break  down^and  give  rise  to  a  secretion  of  milk,  and  this  stage  of 
development  is  attained  at  the  moment  when  the  fetus  is  approaching 
the  full  term  of  gestation,  and  is  about  to  be  delivered. 

It  has  been  ascertained  that  this  remarkably  exact  synchrony  of  the 
development  of  such  widely  separated  organized  bodies  as  the  fetus 
and  the  mammary  glands  of  the  mother  is  brought  about  by  the  circu- 
lation in  the  blood  of  some  as  yet  unindentified  substance  which  is 
elaborated  by  the  tissues  of  the  Placenta.  If  a  saline  extract  of  the 
placentae  of  rabbits  be  injected  repeatedly  into  the  circulation  of  virgin 
rabbits,  the  mammary  glands  hypertrophy  just  as  they  would  if  the 
animal  were  pregnant,  and  finally  secrete  milk  which  may  be  expressed 
from  the  nipples.  Another  factor,  however,  which  may  possibly 
contribute  to  the  development  of  the  mammary  glands  and  their  secre- 
tion of  milk  is  the  slight  measure  of  hypertrophy  of  the  Pituitary  Gland 
which  invariably  accompanies  pregnancy.  The  boiled  aqueous  extract 
of  the  posterior  lobe  of  the  pituitary  gland  contains  a  nitrogenous  base 
of  unknown  constitution,  designated  Pituitrin,  which  increases  the 
irritability  of  the  muscular  walls  of  the  uterus,  causes  an  increase  in  the 
volume  of  the  urine  and  stimulates  the  secretion  of  milk,  the  latter 
effect  being  a  very  unusual  one  for  any  pharmacological  agent  to  bring 
about.  The  large  and  repeated  dosages  of  placental  extract  which 
Starling  and  Lane-Claypon  found  to  be  necessary  to  bring  about  the 
degree  of  hypertrophy  of  the  mammary  glands  which  is  requisite  for  the 
production  of  milk  may  possibly  have  been  attributable  to  the  absence 
of  the  assistance,  in  these  experiments,  which  is  afforded  in  actual 
pregnancy  by  the  enhanced  activities  of  the  pituitary  body. 

That  the  hyperdevelopment  of  the  mammary  glands  of  the  mother 
is  due  to  the  presence  of  stimulators  circulating  in  the  blood,  and  not 
to  any  reflex  nervous  stimulation  of  the  glandular  tissues,  is  shown,  not 
only  by  the  above-cited  experiments,  but  also  by  the  fact  that  the  effect 
of  these  substances  is  not  confined  to  the  mother,  but  extends  to  the 
embryo,  which  is  not  connected  by  any  nervous  channels  with  the 
tissues  of  the  mother.  It  is  a  familiar  fact  that  the  breasts  of  newborn 
infants  frequently  secrete  a  few  drops  of  milk  or  may  be  made  to  do  so 
by  brief  manipulations  of  the  nipples.  The  milk  thus  obtained  was 
known  in  former  days  as  "witches'  milk"  and  was  accredited  by  the 
lady  practioners  of  a  hundred  years  ago  with  many  important  proper- 
ties of  a  supernatural  description. 

When  the  development  of  the  embryo  has  reached  a  certain  stage, 
Uterine  Contractions  bring  about  the  expulsion  of  the  fetus.  We  have 
here  another  example  of  curiously  exact  coincidence  in  time.  It  is  not 
a  question  of  the  size  of  the  developing  fetus  ultimately  bringing  about 
such  a  degree  of  distention  of  the  uterus  as  to  induce  a  special  tendency 
to  contraction,  for  even  the  same  individual  may  deliver  infants  in 


380  EXAMPLES  OF  CHEMICAL  CORRELATION 

successive  births  of  very  varying  size  in  proportion  to  the  bodily  dimen- 
sions of  the  mother.  The  moment  of  delivery  is,  in  fact,. primarily 
determined  by  physiological  factors  in  the  mother,  rather  than  by  the 
stage  of  development  of  the  fetus  at  term.  This  may  be  very  clearly 
seen  by  comparing  the  Variability  of  the  duration  of  gestation  with 
the  variability  of  the  weights  of  the  infants  which  are  delivered. 

The  ordinary  method  of  measuring  the  variability  of  any  quantity 
which  is  adopted  by  statisticians  consists  in  expressing  it  in  terms  of  the 
percentage  ratio  of  the  Standard  Deviation  of  the  quantity  measured  to 
its  average  value.  The  standard  deviation  is  the  square  root  of  the 
mean  square  of  the  observed  deviations  from  the  average.  Thus, 
consider  the  following  illustrative  sets  of  measurements. 

i 11  101 

2 12  102 

3 13  103 

4 14  104 

5 15  105 

It  is  obvious  at  a  glance  that  the  figures  in  the  first  column  are  very 
variable,  those  in  the  second  column  moderately  so,  and  those  in  the 
third  volumn  relatively  invariable  or  approximately  constant.  When 
we  wish  to  express  this  impression  in  arithmetical  terms  we  proceed  as 
follows : 

Average  of  the  first  Average  of  the  second  Average  of  the  third 

column.  column.  column. 

3 .          13  103 

the  deviations  from  the  average  are  in  each  case  2,  1,  0, 1  and  2.  The 
sum  of  the  squares  of  these  deviations  is  4+1+0+1+4=10.  The 
mean  square  is  therefore  2  and  its  square-root,  which  is  the  standard 
deviation,  is  1.414.  The  variability  of  each  of  the  columns  of  figures 
is  the  ratio  of  this  quantity  to  the  average,  expressed  as  a  percentage, 
which  works  out  as  follows: 

Variability  of  first  Variability  of  second  Variability  of  third 

column.  column.  column. 

47. 1  per  cent.  10.9  per  cent.  1 . 37  per  cent. 

Our  impression  of  the  relative  variability  of  the  three  columns  of 
figures  is  thus  expressed  in  quantitative  terms,  the  actual  meaning  of 
the  results  being,  that  in  the  first  set  of  figures  two-thirds1  of  the 
recorded  values  will  be  found  to  differ  by  less  than  47.1  per  cent,  from 
the  mean,  in  the  second  set  two-thirds  of  the  recorded  values  will  differ 
from  the  mean  by  less  than  10.9  per  cent,  of  its  value,  and  in  the  third 
set  of  recorded  values  two-thirds  will  fall  within  1.37  per  cent,  of  the 
mean. 

Applying  this  method  to  the  study  of  the  comparative  variabilities 
of  the  period  of  gestation  and  of  the  weights  of  the  infants  delivered 
thereafter,  we  find  that  the  two  variabilities  bear  no  proportion  to 

1  Or,  more  precisely,  68.27  per  cent. 


CHEMICAL  REGULATION  OF  METABOLISM  381 

one  another,  for  while  the  variability  of  the  weight  of  newborn  infants 
is  14  per  cent.,  that  of  the  length  of  the  period  of  gestation  is  only  4 
per  cent.  There  can  be  little  influence  exerted  by  the  size  of  the  fetus 
upon  the  length  of  gestation,  therefore,  for  otherwise  the  variability 
of  the  period  of  gestation  would  be  nearly  as  great  as  the  variability  of 
the  size  of  the  infants  delivered.  It  is  evident  that  heavy  infants  are 
carried  in  utero  for  a  longer  period  and  light  infants  for  a  snorter  period 
than  wrould  correspond  to  their  relative  development. 

We  must  therefore  look  to  maternal  rather  than  to  fetal  events  for 
the  source  of  the  determination  of  the  period  of  gestation.  Now  the 
investigation  of  the  physiological  condition  of  the  mother  yields  indica- 
tions of  two  factors  which,  as  the  term  of  pregnancy  approaches,  must 
enhance  the  muscular  irritability  of  the  uterus.  The  first  is  the  hyper- 
trophy of  the  Pituitary  Gland,  to  which  reference  has  been  made  above. 
The  aqueous  extract  of  the  posterior  lobe  exerts  a  very  marked  effect 
upon  the  excised  uteri  of  animals,  inducing  powerful  contractions, 
especially  in  the  pregnant  uterus.  The  active  constituent  is  related  to 
but  not  identical  with  /3-Iminazolyl  Ethylamine.  We  may  assume  with 
probability  that  the  hypertrophy  of  the  gland  which  accompanies 
pregnancy  may  result  in  the  presence  of  this  substance  in  increasing 
amounts  in  the  blood-stream  until,  finally,  the  hyperirritability  of  the 
uterus,  with  the  assistance  of  the  second  active  substance  about  to  be 
noted,  reaches  a  stage  culminating  in  contractions  which  expel  the  fetus. 

The  second  factor  which  operates  in  the  direction  of  promoting 
contraction  of  the  uterus,  is  the  presence  of  a  substance  io  the  Colostrum 
or  first  secretion  of  milk,  which  causes  contractions  of  the  pregnant 
uterus.  In  fact  abortion  has  been  brought  about  in  pregnant  cattle 
before  the  normal  period  of  delivery,  by  injections  of  colostrum  from  a 
normal  cow.  Colostrum  differs  in  many  respects  from  the  milk  which 
is  subsequently  secreted.  This  will  be  clear  from  the  following  analyses 
of  cows'  milk,  by  Konig. 

Per  1000.                     Water.           Solids.         Casein.          Other  Fats.  Sugar.  Salts. 

protein. 

Colostrum       .      .      746.7         253.3         40.4         136.0  35.9  26.7  15.6 

Milk     ....     871.7         128.3         30.2             5.3  36.9  48.8  7.1 

It  has  been  recognized  from  a  remote  period  that  colostrum  has  a 
cathartic  action  upon  the  infant,  so  that  the  substance  inducing 
uterine  contractions  may  possibly  be  a  general  muscular  stimulant. 
Its  chemical  nature  is,  however,  unknown. 

THE  CHEMICAL  REGULATION  OF  METABOLISM. 

The  activities  of  our  various  tissues  are  so  closely  interwoven  with 
one  another,  and  the  various  organs  of  the  body  are  so  intimately 
dependent  upon  one  another  for  the  raw  materials  which  they  elaborate 
into  finished  products,  or  the  disposal  of  waste  products  which  might 
otherwise  be  deleterious  to  the  well-being  of  the  whole  bodily  economy, 


382  EXAMPLES  OF  CHEMICAL  CORRELATION 

that  the  complete  analysis  of  the  coordinate  factors  of  our  total  metab- 
olism would  involve  a  survey,  necessarily  incomplete  at  the  present 
stage  of  our  knowledge,  of  the  whole  gamut  of  physiological  activities. 
Without  attempting  to  embark  upon  such  an  ambitious  review,  there 
are  certain  outstanding  factors  in  the  regulation  of  metabolic  activity 
which  compel  our  attention  here,  because  the  regulatory  action  which 
they  exert  would  appear  to  constitute  the  prime  function  of  the  tissues 
concerned. 


FIG.  23. — Cachexia  strumipriva  following  total  extirpation  of  thyroid;  eleven  years 
after  operation.     (After  Kocher.) 

The  most  striking  effects  upon  the  general  metabolism  of  the  body  are 
those  which  are  exerted  by  the  tissues  of  the  Thyroid.  Our  attention 
was  first  drawn  to  the  importance  of  this  gland  in  the  bodily  economy 
by  pathological  conditions  which  are  endemic  in  certain  localities  and 
sporadic  in  all  human  communities.  The  disorders  resulting  from 
improper  functioning  of  the  thyroid  fall  into  two  main  classes,  those 
namely,  which  result  from  subnormal  development  or  activity  of  the 
gland,  and  those  which  result  from  its  overactivity. 

The  condition  of  Myxedema  arises  when  the  thyroid  fails  to  develop 


CHEMICAL  REGULATION  OF  METABOLISM  383 

V 

properly,  or,  in  later  life,  is  extirpated,  or  injured  by  degenerative 
changes.  If  the  failure  of  the  gland  occurs  in  childhood,  intellectual 
development  is  arrested,  and  the  condition  known  as  Cretinism  super- 
venes. The  expression  is  idiotic,  the  skin  is  greatly  thickened  through 
the  overdevelopment  of  connective  tissue,  and  the  features  are  conse- 
quently coarsened  and  brutalized.  In  adults,  extirpation  or  destruc- 
tion of  the  gland  by  disease  results  in  similar  symptoms  (Figs.  23  and 
24)  but  the  intelligence,  although  it  becomes  very  sluggish,  remains 


FIG.  24. — The  same  patient  as  in  Fig.  23,  five  months  after  thyroid  administration. 

(After  Kocher.) 

far  above  the  level  of  an  idiot.  The  temperature  of  the  body  is  sub- 
normal, the  total  metabolism  is  much  reduced,  and  the  daily  nitrogen 
output  is  subnormal.  These  conditions,  if  taken  in  hand  early,  are 
completely  curable  by  the  administration  of  extracts  or  dried  prepara- 
tions of  the  thyroid  gland.  This  is,  in  fact,  the  most  completely  success- 
ful instance  of  organotherapy  to  which  we  are  as  yet  able  to  point,  and 
provided  the  administration  of  the  glandular  preparations  in  appro- 
priate dosage  be  continued,  individuals  who  would  otherwise  exhibit 
most  extreme  symptoms  of  the  disorder  remain  in  satisfactory  health, 
with  unimpaired  intelligence  and  vigor. 


384  EXAMPLES  OF  CHEMICAL  CORRELATION 

The  active  and  remedial  constituent  of  the  gland  is  associated  with 
the  Iodine  which  the  thyroid  contains  and  which  distinguishes  it 
chemically  from  all  other  tissues  of  the  body.1  While  the  iodine-con- 
tent of  the  thyroid  varies  very  much,  not  only  in  different  species  of 
animals,  but  in  different  individuals  of  the  same  species,  yet  the  minimal 
content  of  iodine  which  is  consistent  with  normal  functioning  of  the 
gland  is  very  nearly  constant  and,  on  the  other  hand,  the  remedial 
value  of  a  thyroid  preparation  tends  to  be  proportionate  to  its  iodine- 
content.  The  nature  of  the  active  iodine  compound  has  been  the 
subject  of  very  many  and  extensive  investigations.  The  experiments 
of  Oswald  showed  that  the  active  substance,  as  it  exists  in  the  glandular 
tissue,  is  either  an  iodized  protein  or  closely  associated  with  a  protein 
which  he  termed  Thyreoglobulin.  Bauman  found,  however,  that  the 
partial  hydrolysis  of  Oswald's  thyreoglobulin  by  means  of  sulphuric 
acid  did  not  destroy  its  therapeutic  activity,  but  that  a  fraction  of  the 
hydrolytic  cleavage -products  which  he  termed  lodothyrin  retains  the 
original  activity  of  the  thyreoglobulin.  This  substance,  according  to 
von  Fiirth,  is  related  to  the  "humin"  substances  which  form  in  acid 
hydrolyses  of  protein  in  the  presence  of  carbohydrate  radicals,  and  are 
considered  by  Gortner  and  Blish  to  arise  from  the  Tryptophane  groups 
of  the  protein  molecule.  This  fact  has  received  peculiar  significance 
as  a  result  of  the  recent  researches  of  E.  C.  Kendall  who  has  succeeded 
in  still  further  fractionating  the  hydrolytic  cleavage-products  of 
thyreoglobulin  without  destroying  its  therapeutic  activity.  By 
hydrolyzing  thyreoglobulin  in  alkaline  alcohol  two  groups  of  products 
are  obtained.  The  one  group  is  insoluble  in  dilute  acids,  the  other  is 
soluble.  The  acid-soluble  substances  are  physiologically  and  thera- 
peutically  inert  and  they  contain  very  little  iodine.  The  acid-insoluble 
substances  contain  a  high  proportion  of  iodine,  and  are  physiologically 
and  therapeutically  potent.  By  further  fractionation  Kendall  obtained 
a  white  crystalline  product  containing  60  per  cent,  of  iodine,  which  was 
very  active  therapeutically  and  proved  to  be  a  derivative  of  Indol, 
being  therefore  related  to  tryptophane.  Kendall  believes  that  this 
compound  which  he  designates  Thyroxin  is  a  tri-iodo-oxy-indol-pro- 
pionic  acid,  and  has  tentatively  suggested  the  following  constitutional 
formula: 

HI 


IHC  C  =  =C.CH2.CH2.COOH 

IHC  C  C=O 


H 


1  The  alleged  presence  of    iodine  in  the  pituitary  gland  has  not  proved  possible  to 
confirm. 


CHEMICAL  REGULATION  OF  METABOLISM  385 

The  administration  of  an  excess  of  thyroid  tissue  to  animals  or  man 
is  accompanied  by  a  very  marked  acceleration  of  metabolism.  On  a 
normal  mixed  diet  the  total  heat-output  may  be  raised  100  per  cent. 
The  effect  of  this  enhanced  metabolism  is  to  cause  a  reduction  of  weight 
due  to  loss  of  tissue,  and  especially  of  fat,  and  it  is  for  this  reason  that 
thyroid  extract  is  the  chief  and  only  effective  constituent  of  a  variety 
of  Obesity-cures.  Unfortunately,  however,  the  nitrogenous  output  is 
proportionately  increased,  so  that  the  obese  person  loses  not  only  fat, 
but  also  tissue-protein,  which  he  frequently  can  ill  afford  to  spare. 
Furthermore,  distressing  or  even  dangerous  cardiac  symptoms  are 
liable  to  supervene  with  overdoses  of  thyroid  extract,  or  even  with 
moderate  doses  if  the  thyroid  of  the  patient  is  normally  active,  so  that 
the  unrestricted  use  of  thyroid  preparations  by  the  public  is  attended 
by  serious  danger. 

The  stimulation  of  the  destruction  of  nitrogenous  tissue-constituents 
which  follows  the  administration  of  thyroid  is  extremely  striking. 
Thus,  Rhode  and  Stockholm  have  found  that  in  dogs  receiving  only 
sugar  as  a  diet,  so  that  the  nitrogenous  output  was  minimal,  the  output 
was  increased  fifty  per  cent,  by  so  small  an  amount  as  0.10  to  0.15 
grams  of  dried  thyroid  tissue  per  kilogram  body-weight  of  the 
animals.  Arguing  chiefly  from  the  fact  that  his  crystalline  active 
fraction  reacts  with  amino-acids,  combining  with  the  amino-group  and 
liberating  carbonic  acid,  Kendall  has  advanced  the  view  that  the 
thyroid  secretion  catalyzes  the  process  of  Deaminization  of  amino-acids. 
The  power  of  deaminizing  amino-acids  is  known  to  be  shared  by  all 
the  tissues  and  the  stimulating  effect  of  thyroid  extract  is  likewise 
common  to  all  tissues.  The  question  is  an  extremely  difficult  one  to 
decide,  for  when  we  recollect  that  the  proteins  of  the  tissues  stand  in  a 
relation  of  equilibrium  to  the  reserve  amino-acids  which  they  contain, 
and  that  these  in  turn  are  in  equilibrium  with  the  amino-acids  circu- 
lating in  the  blood  it  is  evident  that  anything  tending  to  break  down 
the  amino-acids  which  have  not  yet  become  integral  living  tissue  must 
also  indirectly  lead  to  the  breaking  down  of  tissue-protein,  and  the 
stimulation  of  endogenous  catabolism.  In  support  of  Kendall's 
theory,  however,  may  be  cited  the  facts  that  hyperthyroidism,  as  in 
exophthalmic  goiter,  is  aggravated  by  a  high  protein  diet,  and  that 
the  effects  of  thyroidectomy  are  more  serious  in  carnivorous  than  in 
herbivorous  animals. 

A  remarkable  effect  of  administration  of  thyroid  tissue  to  mice  is 
the  extraordinarily  increased  tolerance  for  Acetonitrile  to  which  it 
leads.  Reid  Hunt  has  found  that  if  0.1  milligrams  of  dried  thyroid 
tissue  be  administered  to  mice  on  ten  successive  days,  they  will  with- 
stand ten  times  the  normal  lethal  dose  of  acetonitrile,  administered 
subcutaneously,  and  indeed  he  proposes  this  enhanced  tolerance  to  a 
specific  substance  as  a  test  for  the  activity  of  various  thyroid  prepara- 
tions. The  significance  of  this  effect  is,  however,  uncertain  because 
it  is  not  universal;  in  fact  in  such  a  closely  allied  animal  as  the  rat, 
25 


386  EXAMPLES  OF  CHEMICAL  CORRELATION 

administration  of  thyroid  tissue,  so  far  from  enhancing  the  tolerance 
for  acetonitrile,  actually  renders  the  animals  more  sensitive  than  usual 
to  intoxication  by  this  poison. 

Hyperthyroidism  occurs  spontaneously  in  the  condition  known  as 
Basedow's  Disease,  or  Exophthalmic  Goiter.  This  condition  is  accom- 
panied by  enlargement  of  the  gland  and  a  marked  increase  of  secreting 
cellular  elements,  the  interspaces  filled  with  colloidal  material  which 
are  characteristic  of  the  structure  of  this  gland  being  much  reduced 
in  size.  There  is  a  greatly  enhanced  metabolism,  the  calorific  output 
being  frequently  twice  the  normal;  there  is  a  slow  progressive  loss  of 
weight,  incoordination  o£  the  heart-beat  (Tachycardia),  the  tempera- 
ture is  supernormal,  the  nervous  system  hyperirritable,  and  the  blood- 
pressure  is  usually  abnormally  high.  The  rate  and  intensity  of  living 
is  in  fact  increased  in  all  its  aspects,  and  frequently  to  a  dangerous 
extent.  The  administration  of  thyroid  preparations,  or  in  fact  of  any 
iodine-containing  substance,  leads  to  a  reduction  of  the  Hyperplasia 
of  the  epithelium  of  the  gland,  and  an  increase  in  the  quantity  of  col- 
loidal material,  that  is,  to  a  return  toward  the  normal  structure.  It  is 
a  question  whether  the  symptoms  of  Basedow's  disease  are  altogether 
attributable  to  hyperfunctioning  of  the  gland.  The  remedial  effects 
of  iodine  would  point  rather  toward  a  deficiency  of  the  iodine-contain- 
ing principle  as  the  origin  of  the  hyperplasia  of  the  secreting  epithelium 
which  characterizes  the  disease.  In  fact  the  iodine  content  of  the 
hyperplastic  gland  may  actually  be  below  normal,  and  a  similar  condi- 
tion may  be  aroused  in  the  residue  by  excision  of  a  considerable  portion 
of  the  gland,  as  if  the  effort  of  a  small  part  of  the  thyroid  tissue  to 
assume  the  functions  of  the  whole  stimulated  a  proliferation  of  the 
epithelial  elements.  On  the  other  hand  it  must  be  recollected  that  a 
deficient  content  of  any  substance  in  a  secreting  gland  does  not  neces- 
sarily mean  that  the  production  of  the  substance  is  diminished;  it 
may  merely  mean  that  its  rate  of  discharge  from  the  gland  is  abnor- 
mally high,  so  that  it  has  no  opportunity  to  accumulate  within  the 
tissues  of  the  gland  itself. 

The  prevalence  of  Myxedema  and  goiter  in  certain  geographical 
areas  and  particularly  in  mountainous  or  hilly  regions,  and  the  com- 
parative rarity  of  such  conditions  elsewhere,  has  led  us  to  ascribe  the 
endemic  forms  of  thyroid  disease,  directly  or  indirectly,  to  localized 
physiographical  or  geological  conditions.  Even  in  the  days  of  Marco 
Polo,  the  prevalence  of  Goiter  was  attributed  to  a  peculiar  quality  of 
the  water  in  the  localities  affected,1  and  this  impression  still  prevails, 
both  in  medical  and  in  lay  circles.  Notwithstanding  the  clue  this 
offered,  however,  it  has  not  yet  proved  possible  to  establish  the  nature 

"  Departing  from  thence  "  (Samarcand)  "  you  enter  the  province  of  Karkan  .  .  . 
The  people  ...  are  in  general  afflicted  with  swellings  in  the  legs  and  tumors  in 
the  throat,  occasioned  by  the  quality  of  the  water  they  drink."  The  "swellings  in  the 
legs"  are  attributable  to  a  nematode  worm,  Filaria  medinensis  of  which  the  "carrier," 
or  intermediate  host,  is  a  minute  fresh-water  crustacean,  Cyclops. 


CHEMICAL  REGULATION  OF  METABOLISM  387 

of  the  abnormality  in  drinking-water  which  causes  disorders  of  the 
thyroid.  It  cannot  even  be  definitely  stated  whether  the  abnormality 
consists  in  the  presence  of  an  infecting  agent,  or  in  a  chemical  compon- 
ent or  its  absence.  The  numerous  circumscribed  and  yet  widely  sepa- 
rated areas  of  endemic  occurrence,  however,  speak  against  the  view 
that  the  disease  is  communicated  by  an  infecting  organism.  The  goiter 
which  occurs  among  fishes  in  hatcheries,  has  been  traced  to  overfeeding 
with  a  high  protein  diet. 

Lying  just  above  the  thyroid,  or,  in  some  animals,  imbedded  in 
the  thyroid  tissue,  are  a  variable  number  (two  pairs  in  man)  of  small 
glands,  known  collectively  as  the  Parathyroids.  Structurally  they  differ 
essentially  from  the  thyroid  and  evidently  they  also  differ  from  the 
thyroid  very  decisively  in  function,  for  their  excision  leads  to  quite  a 
different  sequence  of  events  from  those  which  follow  thyroidectomy. 
The  removal  of  the  parathyroids,  if  complete,  results  in  acute  neuro- 
muscular  symptoms  which  are  collectively  designated  Tetany,  and 
which  resemble  very  closely  a  condition  which  not  infrequently  arises 
spontaneously  in  young  children.  For  a  little  time  succeeding  para- 
thyroidectomy,  no  abnormalities  appear,  but  within  forty-eight  hours 
tremors  are  observed  in  the  extremities,  followed  by  involuntary  con- 
tractions of  more  and  more  muscles  of  the  body  until,  finally,  convul- 
sions supervene,  terminating  after  several  days  in  death.  The  condi- 
tion is  completely  relieved,  according  to  W.  G.  Macallum,  by  the 
administration  of  Calcium  Salts,  and  for  this  reason  it  was  thought 
probable,  for  some  time,  that  the  special  function  of  the  parathyroids 
consists  in  the  regulation  of  the  Calcium  Metabolism.  Many  facts, 
however,  speak  against  this  view.  In  the  first  place  observers  are  not 
agreed  that  the  excision  of  the  parathyroids  leads  to  increased  excre- 
tion of  calcium  or  a  reduction  of  calcium  in  the  blood  and  tissues,  and 
in  the  second  place  other  disturbances  of  metabolism  to  which  attention 
has  been  directed  in  recent  years  offer  a  more  probable  origin  of  the 
neuromuscular  symptoms.  The  remedial  effect  of  calcium  salts  is 
regarded  merely  as  an  example  of  the  general  action  of  calcium  in  reduc- 
ing the  irritability  of  nerve  fibers.  On  the  other  hand  some  disturbance 
of  the  calcium  metabolism  unquestionably  accompanies  parathyroidec- 
tomy,  for  it  has  been  found  by  Erdheim  that  parathyroidectomy  in 
rats  (probably  not  complete)  leads  to  deficient  dentine-formation  in 
the  teeth  of  the  operated  animals,  and  Erdheim  and  Carrel  have  found 
that  callus-formation  in  injured  bones  is  delayed  by  parathyroidectomy. 

The  effect  of  parathyroidectomy  upon  the  nitrogenous  metabolism 
is  very  marked.  The  output  of  Ammonia  is  much  increased,  and  for 
this  reason  Kendall  and  others  have  suggested  that  the  parathyroids 
control  the  transformation  of  ammonium  carbonate  into  Urea,  which 
is  the  normal  end-result  of  the  deaminization  of  amino-acids,  and  occurs 
primarily  in  the  liver.  There  is  a  decided  Alkalosis  or  increased  alka- 
linity of  the  blood  in  parathyroidectomy,  and  the  symptoms  may  be 
alleviated  by  the  injection  of  acids.  On  the  other  hand  it  has  not 


388  EXAMPLES  OF  CHEMICAL  CORRELATION 

proved  possible  to  induce  tetany  by  injections  of  ammonia  or  ammo- 
nium carbonate. 

The  urine  of  children  between  the  ages  of  two  and  fifteen  normally 
contains  Creatine,  which  is  absent  from  the  urine  of  adults,1  and  it  is 
between  these  ages  that  children  are  most  liable  to  develop  symptoms 
of  tetany.  On  the  other  hand  the  content  of  creatine  in  muscular 
tissues  is  definitely  connected  with  their  Tonus  or  degree  of  tonic 
contraction  and  is  increased  by  all  measures  which  increase  tonus.  It 
has  therefore  been  suggested  by  many  observers  that  the  tetany  arising 
from  parathyroidectomy  may  originate  in  a  disturbance  of  the  normal 
metabolism  of  creatine.  In  this  connection  it  is  of  especial  interest 
to  note  that  Landois  and  Maxwell  have  found  that  while  the  gray 
matter  of  the  motor-areas  of  the  cerebral  cortex  is  remarkably  insensi- 
tive to  the  ordinary  chemical  stimuli  which  increase  the  irritability  of 
nerve-fibers  (calcium  precipitants),  it  is  powerfully  stimulated  by 
applications  of  creatine,  with  the  effect  of  inducing  convulsions.  It  has 
not,  however,  proved  possible  to  induce  tetany  in  animals  by  injections 
of  creatine. 

Creatine  is  methyl  guanidine  acetic  acid,  and  is  therefore  related  to 
the  amino-acid,  Arginine,  the  relationship  of  arginine,  methyl  guanidine 
and  creatine  to  one  another  is  shown  by  the  following  formulae: 

/CH3 


,NH.CH2.CH2.CH2.CH.NH2COOH  /NH.CHs  N— CH2COOH 


C=NH  C=NH  C=NH 

\ 

SNH2  \NH2  ^NHz 

Arginine.  Methyl  guanidine.  Creatine. 

Methylguanidine  and  Dimethyl  Guanidine  occur  in  small  amounts  in 
blood,  muscular  tissues 'and  urine.  It  has  recently  been  shown  by  N. 
Paton  that  the  quantity  of  methylguanidine  in  the  blood  and  urine  is 
decidedly  increased  after  parathyroidectomy  in  animals,  and  in  the 
spontaneous  tetany  which  occurs  in  children.  The  following  figures  are 
illustrative: 

Guanidine  +  Methylguanidine  in  milligrams  per  liter. 

A.  BLOOD. 
DOGS. 

Normal  Parathyroidectorny. 

.  1 . 00  (average  of  5)  8.7  (average  of  8) 

B.  UBINE. 
DOGS. 

Normal.  Parathyroidectomy. 

LO.  25  (average  of  6)  1.1  (average  of  6) 

CHILDREN. 

Idiopathic  tetany. 
0.12  (average  of  8)  (Average  of  3  cases) 

Active  tetany  .  .  .  .  0.58 
Latent  tetany  .  .  .  .  0.38 
Recovery  .  .  .  .  .  0.12 

1  Occasionally  present  in  the  urine  of  women. 


CHEMICAL  REGULATION  OF   METABOLISM  389 

The  subcutaneous  or  intravenous  injection  of  Guanidine  or  Methyl- 
guanidine  was  found  by  Paton  to  lead  to  marked  symptoms  of  tetany. 
Previous  observers  had  established  the  fact  that  guanidine  causes 
fibrillary  twitchings  of  muscular  tissue  through  stimulation,  followed 
by  paralysis  of  the  myoneural  junctions,  and  Fuhner,  in  1906,  demon- 
strated that  this  action  is  antagonized  by  calcium  salts.  The  origin 
of  parathyroid  tetany  would  therefore  appear  to  reside  in  a  disturbance 
of  nitrogenous  metabolism,  and  especially  in  the  metabolism  of  the 
guanidine  derivatives.  The  aggravation  of  symptoms  which  accom- 
panies the  administration  of  a  high  meat-diet  is  thus  accounted  for. 
Whether  the  parathyroids  control  the  metabolism  of  other  nitrogenous 
constituents  of  the  diet  besides  those  which  contain  a  guanidine  nucleus, 
is  unknown,  but  the  alkalosis  which  accompanies  parathyroidectomy 
suggests  that  the  products  of  metabolism  which  the  parathyroids 
remove  or  elaborate  are  strongly  basic  substances  such  as  might  be 
derivable  from  the  decomposition  of  Diamino  Acids,  of  which,  of  course, 
arginine  is  an  example. 

It  has  recently  been  shown  by  Uhlenhuth  that  tetany  may  be  induced 
in  amphibian  larvae  which  do  not  possess  parathyroids  (Amblystoma) ; 
by  the  administration  of  thymus  tissue,  and  he  suggests  that  the  func- 
tion of  the  parathyroids  is  to  remove  or  render  non-toxic  substances 
produced  by  the  Thymus.  This  would  also  explain  the  prevalence  of 
tetany  in  children,  since  the  thymus  degenerates  as  maturity  is  attained. 
While  this  is  very  possible,  it  must  also  be  remembered  that  the  thymus 
is  unusually  rich,  among  animal  tissues,  in  Thymus  Nucleic  Acid,  which 
yields  Guanine  among  its  decomposition-products.  Now  guanine, 
when  oxidized,  yields,  among  other  products,  guanidine,  so  that  the 
tetany  observed  by  Uhlenhuth  may  have  had  a  dietary  rather  than  a 
specific  glandular  origin. 

REFERENCES. 

GENERAL: 

Biedl:     The  Internal  Secretory  Organs,  their  Physiology  and  Pathology,  trans., 
Forster,  London,  1913. 

Swale,  Vincent:     Internal  Section  and  Ductless  Glands,  London,  1912. 

Schajer:     The  Endocrine  Organs,  London,  1916. 

Paton:     The  Nervous  and  Chemical  Regulators  of  Metabolism,  London,  1913. 

Starling:     The  Principles  of  Human  Physiology,  Philadelphia,  1915. 
RESPIRATION: 

Pembrey:     Respiratory  Exchange,  Recent  Advances  in  Physiology  and  Biochem- 
istry, by  Leonard  Hill,  London,  1906. 

Robertson:     Biochem.  Zeit.  Festband  fur  H.   J.  Hamburger,   1908,  p.  287.     Arch. 
Int.  de  Physiol.,  1908,  6,  p.  388.     Pfliiger's  Arch.,  1912,  145,  p.  329. 

Winterstein:     Pfliiger's  Arch.,- 1911,  138,  pp.  159  and  167. 

Laqueur  and  Verzar:     Ibid.,  1912,  143,  p.  395. 

Douglas,  Haldane,  Henderson  and  Schneider:     Phil.  Trans.  Roy.  Soc.,    1913,  203B, 
p.  185. 

Douglas:     Ergeb.  d   Physio!.,  1914,  14,  p.  338. 

Scott:     Am.  Jour.  Physiol.,  1917,  44,  p.  196. 
CIRCULATION: 

Stewart;     Jour.  Exp    Med  ,  1912,  15,  p.  547. 

MacLeod  and  Pearce:     Am.  Jour.  Physiol.,  1911-12,  29,  p.  419. 


390  EXAMPLES  OF  CHEMICAL  CORRELATION 

CIRCULATION  : 

Folin,  Cannon  arid  Denis:     Jour.  BioJ.  Chem.,  1912-13,  13,  p.  477. 

Cannon  and  Gray:     Am.  Jour.  Physiol.,  1914,  34.  p.  232. 

Cannon,  Gray  and  Mendenhall:     Ibid.,  1914,  34,  pp.  243  and  251. 

Cannon:     Bodily  Changes  in  Hunger,  Fear  and  Rage,  New  York,  1915. 

Cannon  and  Caltell:     Am.  Jour.  Physiol.,  1916,  41,  p.  74. 

Stewart  and  Rogoff:     Jour.  Lab.  and  Clin.  Med.,  1918,  3,  p.  209. 
DIGESTION: 

Starling:     Recent  Advances  in  the  Physiology  of  Digestion,  Chicago,  1907. 

Cannon:    Am.  Jour.   Physiol.,    1907-8,   20,   p.    283.     The   Mechanical  Factors  of 
Digestion,  London,   1911. 

Pawlow:     The  Work  of  the  Digestive  Glands,  London,  2d  ed.,  1910. 
ORGANS  OF  GENERATION: 

Lane-Claypon:     Jour.  Physiol.,  1905,  32,  p.  xli. 

Blair-Bell:     British  Med.  Jour.,  1909,  1,  pp.  517  and  592;  1913,  1,  p.  652. 

Marshall:     Physiology  of  Reproduction,  London,  1910. 

Steinach:     Centr.  f.  Physiol.,  1910,  24,  p.  551;  1913,  27,  p.  717.     Pfliiger's  Arch., 
1912,  144,  p.  71. 

Loeb,  L.:     Proc.  Soc.  Exp.  Biol.  and  Med.,  1910,  7,  p.  90.     Jour.  Morphology,  1911, 
22,  p.  37.     Biol.  Bull.,  1914,  27,  p.  1. 

Godlewski:     Physiology  der  Zeugung  in  Winterstein's  Handbuch  de  r  vergleichenden 
Physiologie,  Jena,  1914,  vol.  3,  Pt.  2. 

Robertson:     Am.  Jour,  of  Obstetrics,  1915,  71,  p.  916. 

Lillie,  F.:     Science  N.  S.,  1916,  43,  p.  611;  1917,  23,  p.  371. 
METABOLISM: 

Maxwell:     Jour  Biol.  Chem.,  1906-7,  2,  p.  183. 

Macallum  and  Voegtlin:     Jour.  Exp.  Med.,  1909,  11,  p.  118. 

Kocher:     Les  Prix  Nobel  en  1909,  Stockholm,  1910. 

Cooke:     Jour.  Exp.  Med.,  1910,  12,  p.  45. 

Kendall:     Jour.  Biol.  Chem.,  1915,  20,  p.  501.     Jour.  Am.  Med.  Assn.,  1915,  64,  p. 
2042;  1916,  66,  p.  811.     Endocrinology,  1918,  2,  p.  81. 

Wilson  and  Kendall:     Am.  Jour.  Med.  Sc.,  1916,  151,  p.  79. 

Paton  and  Findlay:     Quar.  Jour.  Exp.  Physiol.,    1916-17,  10,  p.   324. 

Rhode  and  Stockholm:     Jour.  Biol.  Chem.,  1918,  37,  p.  305. 


PART  IV. 

THE  CHEMICAL  PROCESSES  WHICH  UNDER 
LIE  AND  ACCOMPANY  LIFE-PHENOMENA. 


CHAPTER  XVII. 
PROCESSES  INFERRED  FROM  DIRECT  OBSERVATION. 

THE  INTERMEDIATE  METABOLISM  OF  THE  CARBOHYDRATES: 
MUSCULAR  CONTRACTION. 

We  have  seen,  in  considering  the  chemical  regulation  of  the  respira- 
tory movements,  that  the  energy-expenditure  in  muscular  exertion  is 
derived  from  oxidations.  This  follows  immediately  from  the  low  heat- 
value  of  the  hydrolyses  which  occur  in  the  body,  and  which  render 
them  insufficient  sources  of  energy,  and  from  the  greatly  increased 
consumption  of  oxygen  and  output  of  carbon  dioxide  which  accompanies 
the  performance  of  muscular  work. 

It  remains  to  consider,  however,  what  class  of  foodstuffs  undergoes 
the  oxidations  which  release  muscular  energy.  That  Carbohydrates 
afford  a  proportion  of  the  necessary  heat-units  is  evident  from  the  fact 
that  during  the  performance  of  muscular  work  the  Glycogen  which  is 
normally  stored  up  in  muscular  tissues,  is  greatly  diminished  in  quan- 
tity, and  even  the  further  reserve  which  is  stored  up  in  the  liver  becomes 
much  reduced  by  the  performance  of  severe  and  long-sustained  muscu- 
lar work.  The  potential  energy  contained  in  these  reserves  of  glycogen 
is  very  considerable.  Thus  the  liver  of  a  man,  when  fully  stocked 
with  glycogen,  contains  about  150  grams  of  this  polysaccharide, 
while  the  muscles,  at  rest  and  after  feeding,  contain  a  like  amount. 
The  total  available  reserve  of  carbohydrate  material  in  the  body  is 
therefore  about  300  grams,  having  a  heat- value  of  4.1  calories  per 
gram  or  1230  in  all.  If  only  one-fifth  of  this  potential  energy  were 
converted  into  -mechanical  work,  its  remainder  being  dissipated  as 
heat,  it  would  lift  a  weight  of  one  hundred  tons  to  a  height  of  over  three 
feet.1 

The  exhaustion  of  glycogen  by  the  performance  of  Muscular  Work 

1  The  equivalent  of  one  calorie  in  mechanical  work 'is  426.5  kilogram-meters. 


392      PROCESSES  INFERRED  FROM  DIRECT  OBSERVATION 

may  be  observed  in  a  variety  of  ways.  In  the  first  place  we  may 
excise  the  two  corresponding  leg-muscles  of  a  frog,  analyze  one  to  serve 
as  a  standard  for  resting  muscle,  and  stimulate  the  other  with  a  tetaniz- 
ing  current  until  exhaustion  supervenes,  and  the  muscle  will  contract 
no  longer,  and  then  repeat  the  analysis  upon  this  exhausted  muscle. 
The  content  of  glycogen  in  the  stimulated  muscle  is  invariably  found 
to  be  lower  than  in  the  resting  muscle,  as  much  as  fifty  per  cent,  of  the 
glycogen  being  generally  found  to  have  disappeared. 

Another  way  of  approaching  the  problem  is  to  cut  the  motor-nerves 
supplying  one  set  of  leg-muscles  and,  after  the  lapse  of  a  definite  period, 
to  compare  the  glycogen-content  of  these  muscles  deprived  of  nervous 
connections  with  the  glycogen-content  of  the  corresponding  normal 
muscles  on  the  other  side  of  the  body.  The  muscles  of  our  skeleton, 
while  their  nervous  connections  remain  intact,  are  in  receipt  of  con- 
stant slight  nervous  stimuli,  insufficient  to  elicit  actual  contractions, 
but  maintaining  a  condition  of  Tonus  or  constant  tension  which  is  a 
favorable  precedent  to  rapid  and  forcible  movements.  This  tonic 
contraction  of  the  muscles  of  the  skeleton  consumes  energy,  not  in  the 
performance  of  external  work,  it  is  true,  but  in  the  performance  of 
Internal  Work;  the  overcoming  of  resistances  analogous  to  friction  or  to 
the  resistance  to  extension  which  is  displayed  by  a  liquid  surface. 
This  tonus  and  its  resultant  expenditure  of  energy  are  prevented  by 
cutting  off  the  stimuli  which  maintain  it,  so  that  a  muscle  with  its 
motor-nerves  severed,  relaxes,  and  consumes  less  energy  than  a  normal 
muscle  with  its  nervous  connections  intact.  Corresponding  with  this 
we  find  that  the  glycogen  reserves  in  the  paralyzed  muscles  tend  to 
accumulate  and  to  exceed  the  glycogen-content  of  the  innervated 
muscles  on  the  opposite,  unoperated  side  of  the  body.  This  is  clearly 
shown  by  the  following  determinations  by  Marcuse  upon  rabbits,  the 
sciatic  and  crural  nerves  having  been  severed  upon  one  side: 

.  Percentage  of  glycogen. 

Experiment  Paralysed  Innervated 

Number  muscles.  muscle. 

1 0.748  0.539 

2 0.749  0.461 

3 0.589  0.395 

4 0.542  0.341 


Average 0.657  0.434 

The  glycogen-reserve,  through  lack  of  expenditure,  was  therefore 
increased  fifty  per  cent,  in  the  paralyzed  and  demobilized  muscles. 

Again,  we  may  compare  the  glycogen-content  of  all  the  tissues  in 
two  similar  animals,  in  the  one  after  a  period  of  rest,  and  in  the  other 
after  a  period  of  intense  muscular  exertion,  and  we  obtain  again  the 
same  result,  namely  a  disappearance  of  glycogen  with  the  performance 
of  muscular  work.  Thus  Kiilz  forced  a  large  and  well-fed  dog,  weighing 
45.5  kilos,  to  draw  a  heavy  cart  for  nine  hours  and  forty  minutes.  The 
animal  was  then  killed,  and  the  total  glycogen-content  of  all  its  tissues 


INTERMEDIATE  METABOLISM  OF  CARBOHYDRATES      393 

was  determined.  Fifty-two  grams  of  glycogen  were  obtained,  corre- 
sponding to  1.16  grams  of  glycogen  per  kilogram  of  body-weight. 
A  normal  well-fed  dog  of  similar  dimensions  contained  3.8  grams  of 
glycogen  per  kilogram  of  body-weight.  Even  after  four  weeks  of 
starvation  a  similar  dog  was  found  to  contain  1.5  grams  of  glycogen 
per  kilogram  of  its  body-weight,  so  that  somewhat  less  than  ten  hours 
of  severe  muscular  exertion  reduced  the  glycogen  reserves  of  the  body 
to  a  greater  extent  than  four  weeks  of  sheer  starvation. 

So  far,  then,  we  have  proved  that  muscular  energy  may  be  and  is 
derived,  in  part  at  least,  from  the  consumption  of  carbohydrate  mate- 
rials. The  question  now  remains,  what  proportion  of  the  energy  of  muscu- 
lar work  is  provided  by  the  carbohydrates  of  the  food?  For  while  the 
experiments  which  we  have  cited  show  that  a  part,  and  probably  a 
large  part  of  the  energy  expended  in  muscular  work  is  certainly  derived 
from  carbohydrates,  they  do  not  preclude  the  possibility  that  an 
important  proportion  of  the  necessary  heat-units  may  be  supplied  by 
other  foodstuffs,  for  example  by  Proteins. 

This  question  was  answered  as  early  as  1865  by  a  classical  experiment 
which  was  performed  by  Fick  and  Wislicenus.  These  observers 
ascended  Mount  Faulhorn,  climbing  to  a  height  of  1956  meters  above 
the  starting-point.  For  seventeen  hours  before  they  started,  during 
the  six  hours  occupied  in  the  ascent,  and  for  six  hours  following  the 
completion  of  the  ascent  they  consumed  no  food  which  contained 
nitrogen.  The  urine  passed  during  the  ascent  and  in  the  six  hours 
succeeding  the  ascent  was  collected  and  from  its  nitrogen-content  the 
total  quantity  of  body-protein  which  had  been  decomposed  was 
estimated.  It  was  found  that  Fick  had  decomposed  38.3  grams  of 
protein  while  Wislicenus  had  decomposed  37.0  grams.  Now  if  we 
assume,  which,  of  course  is  not  the  fact,  that  all  of  the  protein  was 
decomposed  so  completely  as  to  produce  the  end-products  of  perfect 
combustion,  namely  CO2,  H2O  and  nitrogen,  this  quantity  of  protein 
would  have  liberated  250  calories,  equivalent,  if  it  were  wholly  con- 
verted into  mechanical  work,  to  106,000  kilogram-meters.  But  Wisli- 
cenus, for  example,  weighed  76  kilograms,  and  the  work  which  he 
actually  performed  in  the  mere  effort  of  raising  his  body  through 
1956  metres  was  76X1956=148,656  kilogram-meters,  so  that  upon 
the  most  excessively  liberal  computation  the  protein  which  was  decom- 
posed during  and  after  the  ascent  could  not  possibly  have  furnished 
the  energy  consumed  in  the  ascent.  As  a  matter  of  fact,  the  actual 
yield  of  calories  when  protein  is  burnt  in  the  body  is  much  less  than 
that  which  would  be  derived  if  combustion  were  complete,  for  instead 
of  nitrogen  being  formed  the  oxidation  stops  with  the  production  of 
Urea  which  has  a  very  considerable  heat-value  of  its  own  and  which 
is  voided  from  the  body  and  not  utilized.  Furthermore  no  machine  is 
known,  not  even  a  living  machine,  which  can  quantitatively  convert 
heat  into  mechanical  work.  In  fact  actual  measurements  have  shown 
that  only  twenty  per  cent,  of  the  heat-value  of  foods  is,  as  a  rule, 


394     PROCESSES  INFERRED  FROM  DIRECT  OBSERVATION 


available  for  the  production  of  mechanical  work.  If  we  apply  these 
various  corrections  to  the  above 'estimate  of  the  work  available  from 
the  proteins  destroyed  by  Fick  and  Wislicenus,  we  find  that  it 
actually  amounts  to  only  13,000  kilogram-meters,  or  less  than  nine 
per  cent,  of  the  work  required  merely  to  lift  the  weight  of  their 
bodies  to  the  top  of  the  mountain.  Now  it  must  be  remembered 
that  the  ascent  of  their  bodies  was  by  no  means  the  whole  of 
the  mechanical  work  which  was  performed  by  these  experimenters, 
for  apart  from  the  tonus  of  their  skeletal  muscles,  the  work  of  the 
secretory  and  excretory  organs,  and  the  movements  of  the  digestive 
canal,  expenditures  of  energy  that  cannot  very  easily  be  computed, 
their  circulations  had  to  be  maintained  by  the  beating  of  their  hearts 
and  their  respiratory  movements  by  contractions  of  the  diaphragm  and 
intercostal  muscles.  These  sources  of  expenditure  of  energy  alone  can 
be  estimated  to  have  accounted  for  no  less  than  30,000  kilogram-meters 
of  work  during  the  ascent  of  the  mountain.  All  the  energy  actually 
procurable  from  the  protein  they  decomposed,  therefore,  would  not 
have  half  sufficed  to  maintain  the  respiratory  movements  and  the 
heart-beat,  leaving  nothing  over  whatever  for  the  ascent  of  the  moun- 
tain. The  proportion  of  muscular  energy  furnished  by  the  proteins 
must  therefore  have  been  very  small. 

That  under  normal  conditions  the  whole  of  the  energy  consumed  in 
muscular  exertion  is  derived  from  non-protein  sources,  is  rendered 
very  probable  by  the  discovery  of  Voit,  that  work  upon  the  treadmill 
by  a  dog  fed  upon  mixed  rations  does  not  increase  the  nitrogen  output. 
Not  only  is  the  total  nitrogen  output  unaffected  by  muscular  work 
upon  a  mixed  diet,  but  the  entire  Protein  Metabolism  pursues  its  normal 
course,  undisturbed  by  the  large  expenditure  of  energy  which  is  occur- 
ring. This  is  shown  by  an  experiment  by  Shaffer,  who  investigated  the 
urine  of  a  man  fed  upon  a  purine-free  diet,  containing  a  minimal  allow- 
ance of  nitrogen,  in  three  different  periods,  namely,  a  rest-period  of 
six  days  which  he  spent  in  bed;  a  normal  period  of  five  days  which  he 
spent  in  performing  light  work  about  the  laboratory,  and  a  work  period 
of  four  days  in  which  he  added  to  the  laboratory  work  long  daily  walks. 
The  following  were  the  results  obtained : 


Period. 

Food. 

Urine. 

N. 
grams. 

Calories. 

Total 

N. 

Nitrogen  present  as: 

Sulphur. 

Ammonia. 

Creatinin. 

Uric 
acid. 

Undeter- 
mined. 

I.  Rest 
II.  Normal 
III.  Work    . 

5.9 
6.0 
5.9 

2300 
3000 
3200 

4.77 
4.40 
3.94 

0.35 
0.38 
0.42 

0.605 
0.600 
0.560 

0.11 
0.106 
0.12 

0.35 
0.42 
0.42 

0.438 
0.424 
0.414 

The  question  arises,  however,  whether,  if  placed  under  practical 
compulsion  to  do  so,  by  the  scarcity  or  absence  of  other  source  of 


INTERMEDIATE  METABOLISM   OF  CARBOHYDRATES      395 

energy,  the  muscular  tissues  may  not  be  able  to  utilize  proteins  for  the 
performance  of  mechanical  work.  Experiments  of  Kellner,  conducted 
upon  horses,  render  this  very  probable,  for  this  observer  found  that 
while  muscular  work  upon  a  mixed  diet,  as  Voit  had  previously  shown, 
does  not  increase  the  nitrogenous  output  of  the  horse,  yet  muscular 
work  upon  a  diet  which  contained  an  insufficient  allowance  of  carbo- 
hydrates did  result  in  a  notable  increase  of  nitrogen  elimination.  This 
fact  may  be  paralleled  by  the  oft-repeated  observation  that  while 
Bacteria  will  preferably  obtain  their  energy  from  carbohydrates  in  the 
culture-medium,  yet  if  these  be  insufficient  in  amount,  proteins  are 
attacked  and  energy  is  derived  from  the  hydrocarbon  radicals  which 
they  contain,  nitrogenous  fragments  being  split  off  as  by-products  of 
the  process. 

Now  proteins,  being  an  abnormal  source  of  muscular  energy,  may 
very  possibly  give  rise  to  some  unusual  products  when  necessity  com- 
pels their  utilization  for  this  exceptional  purpose.  We  recognize  that 
the  protein  metabolism  of  muscular  tissues  is  peculiar.  The  abundance 
of  Creatine  in  the  muscles  and  the  presence  of  Methylguanidine,  Dimethyl- 
guanidine,  Carnitine  and  other  physiologically  active  nitrogenous  bases 
in  muscular  tissues  show  that  the  degradation  of  protein  in  these 
tissues  does  not  follow  the  channels  normal  to  other  tissues,  and  arouses 
the  suspicion  that  rapid  and  extensive  breaking-down  of  muscle-pro- 
teins might  lead  to  the  production  of  toxic  bases  in  dangerous  amounts 
and  to  notable  physiological  disturbances.  We  are  reminded,  in  this 
connection,  of  the  fact  that  the  dangerous  toxemia  of  pregnancy, 
Eclampsia,  is  often  accompanied  by  sudden  involution  (degeneration) 
of  the  muscular  tissues  of  the  uterus.  Nor  are  there  wanting  facts 
which  tend  directly  to  show  that  extreme  muscular  exhaustion  upon 
a  high  protein  diet  may  be  dangerous.  The  experiences  of  Mawson 
and  Mertz  in  the  Australian  Antarctic  expedition  of  1912-1913,  which 
culminated  in  the  tragic  death  of  Dr.  Xavier  Mertz,  may  be  instanced. 
In  severe  antarctic  weather  and  heavily  crevassed  country,  involving 
extraordinary  expenditures  of  energy  to  maintain  bodily  heat  and  make 
progress  over  the  ground,  at  a  distance  of  three  hundred  miles  from 
headquarters,  these  explorers,  through  loss  of  a  companion  and  a 
sledge  in  a  crevasse,  found  themselves  with  a  bare  one  and  a  half  weeks' 
food  for  themselves,  and  none  at  all  for  the  dogs.  They  started  to 
walk  back  to  their  headquarters,  killing  the  dogs  from  time  to  time  and 
consuming  their  necessarily  excessively  lean  flesh.  After  eighteen 
days  Mertz  began  to  fail,  and  during  several  days  expressed  especial 
aversion  to  the  dogs'  meat;  he  displayed  great  muscular  weakness, 
and  complained  of  Violent  abdominal  pains  from  which  Mawson  also 
suffered.  Seven  days  later  symptoms  of  central  nervous  intoxication 
appeared.  The  following  are  notes  from  Mawson's  diary: 

"January  7. — It  was  a  sad  blow  to  me  to  find  that  Mertz  was  in  a 
weak  state  and  requited  helping  in  and  out  of  his  bag.  He  needed 
rest  for  a  few  hours  at  least  before  he  could  think  of  travelling.  I  have 


396      PROCESSES  INFERRED  FROM  DIRECT  OBSERVATION 

to  turn  in  again  to  kill  time  and  also  to  keep  warm  for  I  feel  the  cold 
very  much  now." 

"At  10  A.M.  I  get  up  to  dress  Xavier  and  prepare  food,  but  find 
him  in  a  kind  of  fit.  Coming  round  a  few  minutes  later,  he  ex- 
changed a  few  words  and  did  not  seem  to  realize  that  anything  had 
happened  .  .  ." 

"During  the  afternoon  he  had  several  more  fits,  then  became  delirious 
and  talked  incoherently  until  midnight,  when  he  appeared  to  fall  off 
into  a  peaceful  slumber.  .  .  After  a  couple  of  hours,  having  felt 
no  movement  from  my  companion,  I  stretched  out  an  arm  and  found 
that  he  was  stiff."1 

These  are  not  symptoms  of  mere  inanition.  Definite  intoxication 
was  also  present,  and  it  appears  not  improbable  that  the  extraordinary 
exertions  necessitated  by  their  situation,  carried  out  as  they  were  upon 
an  almost  exclusively  protein  diet,  may  have  led  to  the  abnormal 
disintegration  of  food-  and  tissue-proteins  by  the  muscular  tissues,  with 
the  production  of  poisonous  nitrogenous,  fragments. 

The  employment  of  a  high  protein  diet  as  a  preparation  for  muscular 
exertion  and  endurance  is  therefore  in  the  highest  degree  irrational, 
more  especially  since  the  rate  of  loss  of  heat  from  the  body  on  a  protein 
diet  is  diminished,  so  that  the  cooling  necessary  for  the  maintenance  of 
prolonged  bodily  effort  is  rendered  more  difficult  than  usual.  The 
only  possible  ground  for  the  formerly  popular  dietary  of  beefsteak  for 
athletes  is  the  fact  that  on  a  diet  purely  of  flesh  the  muscular  machine  is 
more  efficient,  i.  e.,  produces  less  heat  per  unit  of  external  work  per- 
formed. In  fact  in  a  dog  fed  upon  pure  flesh  Pflueger  obtained  the 
highest  work-yield  that  has  ever  been  observed,  nearly  fifty  per  cent, 
of  the  heat-value  of  the  food  appearing  as  mechanical  work.  For  a 
short,  sharp  "dash"  or  brief  effort,  therefore,  a  high  protein  diet  may 
possess  advantages,  but  for  prolonged  extreme  exertion  a  mixed  diet 
containing  an  exceptionally  abundant  allowance  of  carbohydrates  is 
the  only  rational  prescription.  This  is,  in  fact,  the  actual  dietary 
which,  in  the  absence  of  suggestion  or  direction,  is  voluntarily  chosen 
by  those  classes  and  groups  of  individuals  whose  mode  of  earning  a 
living  compels  great  and  sustained  muscular  effort. 

The  normal  source  of  muscular  energy  is  therefore  the  carbohydrates 
of  the  dietary.  That  the  Fats  may  also  be  utilized  for  this  purpose  is 
evidenced  by  the  fact,  first  established  by  Rubner,  that  fat  and  carbo- 
hydrate are  Isodynamic  Foodstuffs,  i.  e.,  that  equicalorific  amounts  of 
these  substances  can  replace  one  another  in  the  diet.  There  has  been 
some  discussion  of  the  question  whether  or  not  the  fats  are  directly 
utilized  for  the  performance  of  work,  or  whether  they  may  not  have 
to  undergo  a  preliminary  transformation  into  carbohydrates.  This 
question  has  been  experimentally  investigated  by  Zuntz,  who  found 
that  when  carbohydrates  predominate  in  the  diet  the  total  amount  of 

1  The  Home  of  the  Blizzard,  Sir  Douglas  Mawson,  London,  1915,  vol.  i,  pp.  258-259. 


INTERMEDIATE  METABOLISM  OF  CARBOHYDRATES      397 

energy  liberated  by  the  body  (work  plus  heat)  corresponds  to  9.33 
small  calories1  for  every  kilogram-meter  of  work  performed,  while  if  the 
carbohydrate  be  replaced  by  fat,  the  total  liberation  of  energy  is  10.37 
calories  for  the  same  amount  and  kind  of  work.  Now  2.35  small 
calories  are  equivalent  to  one  kilogram-meter  of  mechanical  work,  so 
that  on  a  carbohydrate  diet  25  per  cent,  of  the  excess  of  energy-dissipa- 
tion due  to  work  was  actually  converted  into  mechanical  work,  and  on  a 
fat  diet  22.7  per  cent.  There  is  thus  little  difference  of  efficiency 
whether  fats  or  carbohydrates  furnish  the  source  of  energy.  Now  if 
fats  had  first  of  all  to  be  converted  into  carbohydrates,  before  they 
could  be  utilized  for  work,  a  great  deal  of  oxygen  would  have  to  be 
introduced  into  the  molecule,  since  the  fats  contain  a  much  higher  pro- 
portion of  hydrogen  to  oxygen  than  the  carbohydrates.  If  all  this 
preliminary  oxidation  were  unavailable  for  the  production  of  muscular 
energy,  not  less  than  29  per  cent,  of  the  energy  of  the  fat  would  be 
wasted,  and  we  would  expect  the  performance  of  mechanical  work  on  a 
fat  diet  to  be  only  two-thirds  as  efficient  as  upon  a  carbohydrate  diet. 
It  is  highly  probable;  therefore,  that  fats  undergo  but  little  preliminary 
modification  before  they  are  available  for  muscular  work.  They  are 
not  the  first  choice  of  the  muscles,  however,  if  all  dietary  materials  are 
available,  carbohydrates  are  used  first.  Fats  are  pressed  into  the 
service  when  carbohydrates  begin  to  fail,  and  proteins  form  a  last 
resource. 

The  performance  of  muscular  work  involves  a  considerable  increase 
of  oxygen-intake,  and  carbon-dioxide  output.  The  final  products 
of  muscular  exertion  are  therefore  carbon  dioxide  and  water.  The 
oxidation  of  glycogen  or  its  hydrolytic  cleavage-product,  glucose,  is  not 
accomplished  in  a  single  step,  however.  Intermediate  products  are 
transiently  formed,  and  of  the  nature  of  many  of  these  we  can  only 
form  conjectures  which,  however,  are  gradually  becoming  more  and 
more  clearly  defined  as  persistent  research  reveals,  one  after  another, 
the  various  substances  which  may  arise  from  the  oxidations  of  glucose 
in  the  animal  body.  One  of  the  first  of  these  intermediate  products 
to  be  clearly  recognized  was,  however,  Lactic  Acid. 

The  lactic  acid  which  is  found  in  muscular  tissue  is  not  the  ordinary 
racemic  acid  which  may  be  obtained  by  synthesis  in  laboratory-glass- 
ware. It  is  the  dextrorotatory  acid,  or  Sarcolactic  Acid: 

CH3 

I 

CHOH 
I 
COOH 

which,  when  pure,  forms  a  viscous,  acid  syrup,  forming  crystalline  salts 
with  a  variety  of  bases.  The  zinc  salt  is  the  one  usually  employed  for 
the  isolation  and  estimation  of  lactic  acid  in  muscular  tissues. 

1  The  small  calorie  is  the  heat  required  to  raise  the  temperature  of  1  gram  of  water 
one  degree.  The  large  calorie  is  the  heat  required  to  raise  the  temperature  of  a  kilogram 
of  water  one  degree. 


398      PROCESSES  INFERRED  FROM  DIRECT  OBSERVATION 

There  has  been  some  discussion  of  the  question  whether  the  lactic 
acid  of  muscular  tissues  actually  arises  from  the  partial  oxidation  of 
carbohydrates  or  whether  it  may  not,  on  the  contrary  arise  from 
Proteins,  as,  for  example,  by  the  deaminization  of  the  Alanine  radical  of 
proteins: 

CH3  CH3 

I  I 

CHNH2  +     H2O      =     CHOH     +     NH3 
I 
COOH  COOH 

Alanine.  Lactic  acid. 

While  such  an  origin  of  sarcolactic  acid  must  be  admitted  to  be 
possible,  yet  it  is  more  probable  that  the  major  part  of  the  lactic  acid 
produced  by  the  muscular  and  other  tissues  of  the  body,  arises  from 
a  carbohydrate  source.  Thus  Mandel  and  Lusk  have  shown  that  in 
Phosphorus-poisoning  there  is  a  great  increase  in  the  lactic-acid  output 
in  the  urine.  If,  however,  the  body  has  previously  been  drained  of  its 
carbohydrate  reserve  by  inducing  Glycosuria  through  the  administra- 
tion of  Phloridzin,  then  phosphorus-poisoning  results  in  no  hyper- 
production  of  lactic  acid. 

The  lactic  acid  in  excised  muscles  of  the  frog  rapidly  diminishes  on 
standing.  This  is  due  to  its  oxidation  by  the  muscle-tissues.  Now  the 
oxidation  of  lactic  acid  is  evidently  a  more  difficult  step  to  accomplish 
than  its  production  from  glucose  or  glycogen,  for,  if  the  oxygen  supplied 
to  the  muscles  be  interfered  with  by  asphyxia,  by  inhalation  of  air  poor 
in  pxygen,  or  by  poisoning  with  Carbon  Monoxide,  the  lactic-acid  con- 
tent of  the  tissues  and  of  the  blood  and  urine  is  enormously  increased. 

One  of  the  characteristics  of  extreme  muscular  Fatigue  is  the  stiffen- 
ing and  inextensibility  of  the  muscles  which  ensues.  After  death  the 
Rigor  Mortis  or  postmortem  stiffening  of  the  muscles  occurs  with 
extreme  rapidity  if  the  animal  has  immediately  prior  to  death  been 
engaged  in  extreme  and  prolonged  muscular  exertion.  The  stiffening 
and  increased  opacity  of  the  muscles  which  occurs  after  extreme  fatigue 
or  death  is  due  to  the  coagulation  of  certain  proteins  which  the  muscle- 
fluids  contain,  the  semifluid  Muscle-plasma  being  converted  into  a 
jelly. 

It  was  found  by  Halliburton  that  if  muscles  be  frozen  and  minced 
and  then  subjected  to  pressure  at  a  temperature  slightly  above  freezing, 
an  opalescent  fluid  is  obtained  which  clots  spontaneously  upon  warming 
to  a  little  above  bodily  temperatures,  or  upon  standing  for  some  time  at 
room-temperatures.  According  to  von  Fiirth  the  gelatinization  of 
this  fluid  is  due  to  changes  which  occur  in  two  proteins,  the  one  a  globu- 
lin, Myosin  and  the  other  an  albumin,  Myogen.  The  myogen  fraction 
is  much  the  more  abundant  of  the  two.  Upon  heating  or  acidification 
these  soluble  proteins  are  transformed,  respectively,  into  Myosin 
Fibrin  and  Myogen  Fibrin.  The  process  is  not  reversible;  the  jelly 
cannot  be  liquified  by  cooling  or  by  neutralization.  It  is  believed  that 
the  partial  gelatinization  of  these  proteins  which  constitutes  rigor  in 


INTERMEDIATE  METABOLISM  OF  THE  FATS  399 

muscles  is  brought  about  by  the  lactic  acid  and  even,  in  part,  by  the 
carbon  dioxide  which  accumulates  in  fatigued  muscles. 

The  Creatine  content  of  muscular  tissues  is  not  decisively  affected  by 
muscular  work.  It  appears  that  the  increase,  if  any,  is  very  slight,  a 
fact  which  corresponds  to  the  subordinate  part  which  is  normally 
played  by  proteins  in  the  development  of  muscular  energy.  Neverthe- 
less Van  Hoogenhuyze  and  Verploegh  have  found  a  definite  increase 
of  creatine  in  muscular  tissues  after  severe  work,  provided  the  work 
was  performed  by  starving  animals.  In  other  words  if  protein  is  of 
necessity  employed  by  the  muscles  as  a  source  of  energy,  then  creatine 
is  numbered  among  the  chemical  products  of  muscular  work.  The 
production  of  creatine  appears,  however,  to  bear  an  especial  significance 
in  relation  to  muscular  Tonus;  any  agent  tending  to  increase  the  tonic 
contraction  of  the  muscles  leading  to  an  increased  creatine-content. 
Thus  the  creatine-content  of  the  muscles  is  increased  by  drugs  such  as 
Cinchonine  which  increase  tonus,  and  in  pregnancy  the  creatine-content 
of  the  muscular  tissues  of  the  Uterus  is  very  greatly  increased. 


THE  INTERMEDIATE  METABOLISM  OF  THE  FATS;  DIABETES. 

The  normal  products  of  the  oxidation  of  the  fats  and  sugars  are 
finally,  as  we  have  seen,  carbon  dioxide  and  water.  In  animals  with 
normal  metabolism,  but  few  of  the  intermediate  products  of  oxidation 
can  be  perceived,  because  the  various  stages  are  passed  through  rapidly 
when  the  oxidation  is  once  begun,  and  intermediate  products  of  the 
process,  therefore,  have  no  opportunity  to  accumulate.  One  stage 
which  is  easily  recognizable  is  that  afforded  by  the  production  of 
Lactic  Acid  because  the  next  step  in  the  oxidative  processes  is  evidently 
accomplished  with  relative  difficulty,  so  that  a  proportion  of  this  prod- 
uct accumulates  in  the  tissues,  especially  if  the  oxidative  processes 
are  interfered  with  so  as  to  increase  the  difficulty  of  further  transfor- 
mation. Our  knowledge  of  other  stages,  in  the  oxidation-processes 
of  the  body  is,  however,  very  largely  derived  from  an  experiment  which 
is  performed  for  us  by  nature  in  the  disease  or  group  of  diseases  known 
as  Diabetes  Mellitus. 

Glycosuria,  the  excretion  of  sugar  in  the  urine,  may  be  induced  by 
the  injection  of  physiologically  unbalanced  Salt  Solutions  and  particu- 
larly by  solutions  containing  Magnesium  Salts.  The  origin  of  this 
glycosuria,  whether  it  arises  from  an  unusual  discharge  of  sugar  from 
the  muscles  or  the  liver,  or  from  an  increased  permeability  of  the 
kidneys  for  sugar,  has  not  as  yet  been  ascertained.  A  glycosuria 
without  any  accompanying  Glucohemia,  that  is,  without  any  increase 
in  the  normal  percentage  of  sugar  in  the  blood,  may  be  induced  by  the 
administration  of  the  glucoside  Phloridzin.  This  glycosuria  is  evidently 
due  to  an  alteration  of  the  normal  Permeability  of  the  kidney  for  sugar. 
The  epithelium  of  the  normal  kidney  interposes  an  impassable  barrier 


400      PROCESSES  INFERRED  FROM  DIRECT  OBSERVATION 

to  the  passage  of  sugar  into  the  urine  provided  that  the  sugar  in  the 
blood  does  not  much  exceed  the  normal  concentration  of  0.10  to  0.15 
per  cent.  After  treatment  with  phloridzin,  however,  this  barrier  breaks 
down.  The  normal  sugar-content  is  drained  out  of  the  blood,  and  the 
liver  and  muscles,  in  the  endeavor  to  restore  the  normal  equilibrium 
between  Glycogen  and  Glucose,  release  glucose  continuously  to  the 
blood,  so  that  the  ultimate  result  is  the  drainage  of  the  carbohydrate 
reserves  of  the  body.  That  the  effect  is  a  purely  local  one  upon  the 
epithelium  of  the  kidneys  is  shown  by  the  fact  that  if  the  phloridzin 
be  supplied  only  to  one  kidney  by  perfusion  into  the  corresponding 
renal  artery,  that  kidney,  but  not  the  other,  will  eliminate  glucose. 
It  has  been  supposed  that  phloridzin,  being  a  glucoside,  acts  as  a  carrier 
of  glucose  across  the  kidney-epithelium,  liberating  glucose  on  the  one 
side  and  combining  with  it  upon  the  other,  but  of  this  we  have  no 
definite  proof. 

Yet  again,  glycosuria  may  result,  temporarily,  from  an  excessive 
ingestion  of  carbohydrates,  and  particularly  of  sugars.  This  form 
of  glycosuria,  known  as  Alimentary  Glycosuria  is  not  serious  unless, 
indeed,  it  occurs  too  readily,  when  it  may  indicate  a  slight  or  incipient 
diabetes.  It  is  stated  by  Gushing  that  alimentary  glycosuria  tends 
especially  to  occur  in  conditions  of  Hyperpituitarism  or  overactivity  of 
the  pituitary  gland,  of  which  condition,  in  fact,  he  considers  a  readily 
elicited  alimentary  glycosuria  to  afford  confirmatory  diagnosis.  In  the 
opposite  condition  of  Hypopituitarism  he  finds,  on  the  contrary,  an 
extraordinary  tolerance  for  ingested  sugars  and  alimentary  glycosuria 
fails  to  appear 'after  a  dosage  of  glucose  or  levulose  which,  in  normal 
individuals,  would  inevitably  be  followed  by  an  excretion  of  sugar  in 
the  urine.  Other  observers,  while  confirming  Cushing's  observation 
that  pituitary  disease  is  accompanied  by  disturbances  in  the  carbohy- 
drate-tolerance, do  not  concur  with  him  in  his  view  of  the  relationship 
of  the  disturbance  to  hyper-  or  hypo-functioning  of  the  pituitary  gland. 
It  must  be  recollected  in  this  connection,  however,  that  our  means  of 
distinguishing  between  hyper-  and  hypo-activity  of  the  pituitary  gland 
are  rendered  very  imperfect  by  the  fact  that  the  physical  effects  of 
previous  hyperactivity  of  the  pituitary  body  persist,  and  may  in  fact 
constitute  the  most  prominent  symptoms,  long  after  the  condition  has 
passed  into  one  of  deficient  activity  of  the  gland. 

The  possible  involvement  of  the  nervous  system  in  the  etiology  of 
^jt  diabetic  conditions  was  very  strikingly  brought  into  prominence  by  the 
discovery  of  Claude  Bernard  in  1854  that  injury  of  a  certain  area  in 
the  medulla  oblongata  induced  a  transitory  but  severe  glycosuria. 
The  particular  area  concerned  lies  between  the  level  of  the  origins  of 
the  auditory  nerves  and  the  vagi.  The  Diabetic  Puncture  is  most  suc- 
cessful in  animals  that  have  been  well  fed  with  carbohydrates  and  may 
fail  in  ill-nourished  animals.  The  immediate  cause  of  the  excretion  of 
sugar  which  follows  this  operation  is  a  pronounced  Glucohemia,  the 
sugar  in  the  blood  rising  from  the  normal  level  of  0.10  or  0.15  per  cent. 


INTERMEDIATE  METABOLISM  OF  THE  FATS  401 

to  0.3  per  cent,  or  more,  and  the  kidneys  simply  excrete  that  proportion 
of  the  blood-sugar  which  constitutes  an  excess  over  the  normal  amount. 

The  glucohemia  which  ensues  after  the  diabetic  puncture  is  evidently 
due  to  a  failure  of  the  normal  power  of  the  liver  to  store  up  glucose  in 
the  form  of  its  anhydride,  glycogen.  The  efficiency  of  the  operation  is 
proportional  to  the  glycogen-content  of  the  liver  at  the  time  it  is  per- 
formed, and  at  the  end  of  the  process  the  liver  is  found  to  have  been 
drained  of  its  glycogen-reserves.  It  appears  that  the  storage-capacity 
of  the  liver  is  subject  to  control  by  the  nervous  system.  The  afferent 
path  in  the  reflex  arc  is  contained  in  the  vagi.  If  the  vagus  is  cut 
and  the  peripheral  end  is  stimulated  no  glycosuria  ensues,  but  if  the 
central  end  is  stimulated  a  decided  discharge  of  sugar  from  the  liver 
occurs.  The  efferent  paths  lie  in  the  splanchnic  nerves,  and  if  these  be 
previously  severed  the  diabetic  puncture  is  without  effect. 

The  greatest  advance  toward  the  interpretation  of  spontaneous 
diabetes,  however,  occurred  when  in  1889  von  Mering  and  Minkowski 
discovered  that  extirpation  of  the  Pancreas  in  animals  produces  a  pro- 
found glucohemia  and  glycosuria  terminating  ultimately  in  the  death 
of  the  animal.  The  effects  of  this  operation  have  been  very  exhaus- 
tively studied  in  recent  years  by  F.  M.  Allen  who  finds  that  glucohemia 
and  glycosuria  may  be  induced  by  partial  removal  of  the  pancreas. 
If  nine-tenths  of  the  gland  be  excised  a  severe  diabetes  ensues,  but  if 
only  a  small  part  of  the  pancreas,  for  example  one-eighth,  be  removed,  a 
mild  diabetes  ensues  which  is  modifiable  by  diet.  Thus  if  a  sufficiency 
of  the  pancreas  be  left  in  situ  no  glycosuria  at  all  may  appear  in  the 
urine.  If  the  remnant  of  gland  be  larger  glycosuria  may  be  absent 
on  a  meat-diet  or  even  on  a  diet  containing  bread,  but  glycosuria  will 
ensue  if  sugars  be  added  to  the  diet  and,  once  started,  may  continue 
on  a  bread-and-meat  diet.  In  turn,  continued  glycosuria  upon  a  bread- 
and-meat  diet  may  culminate  in  a  condition  in  which  glycosuria  con- 
tinues on  meat  alone,  and  the  experiment  terminates  fatally. 

The  interesting  observation  has  been  made  by  Carlson,  that  if  glyco- 
suria be  induced  in  a  female  animal  by  depancreatization,  and  the 
animal  subsequently  becomes  pregnant,  the  glycosuria  ceases  at  the 
time  that  the  pancreas  begins  to  develop  in  the  embryo.  It  is  not 
certain,  however,  whether  this  is  due  to  the  mother  being  enabled  to 
utilize  glucose  herself  through  transmission  of  a  pancreatic  hormone 
from  the  fetus  to  the  maternal  circulation,  or  whether,  which  is  perhaps 
more  probable,  the  drainage  of  carbohydrates  from  the  mother  by  the 
needs  of  the  fetus  deprives  her  of  the  excess  which  she  is  unable  to 
utilize  herself. 

In  fatal  cases  of  diabetes  it  has  repeatedly  been  observed  that 
degenerative  changes  are  present  in  certain  elements  of  the  pancreatic 
tissues,  namely  the  Islets  of  Langerhans,  and  it  is  particularly  to  the 
removal  of  these  elements  that  the  diabetes  following  total  or  partial 
extirpation  of  the  pancreas  is  due.  Thus  injection  of  paraffin  into 
the  ducts  arising  from  the  secretory  tissues  of  the  pancreas  results  in 
26 


402      PROCESSES  INFERRED  FROM  DIRECT  OBSERVATION 

complete  atrophy  of  the  secreting  epithelium,  the  Islets  of  Langerhans 
alone  remaining  unimpaired.  Under  these  circumstances  no  glycosuria 
occurs,  but  if  this  atrophied  remainder  of  the  gland  be  removed  typical 
pancreatic  diabetes  at  once  occurs.  When  the  pancreas  is  only  parti- 
ally removed  the  overstrain  upon  the  remainder  of  the  tissues  leads  to 
their  degeneration  and  the  symptoms,  possibly  slight  at  first,  become 
progressively  more  severe.  According  to  Allen,  however,  if  the  residue 
of  pancreatic  tissue  be  sufficient  and  overstrain  be  avoided  by  a  diet 
low  in  carbohydrates  and  in  fats,  the  incidence  of  progressive  degenera- 
tive changes  in  the  residual  tissues  may  be  avoided. 

The  occurrence  of  spontaneous  Diabetes  in  human  beings  has  been 
recognized  from  very  ancient  times,  but  the  actual  identification  of  the 
sweet  constituent  of  the  urine  as  Glucose  was  not  accomplished  until 
1838.  It  is  characterized,  it  would  appear,  almost  if  not  quite  invari- 
ably by  a  distinct  Glucohemia.  It  is  improbable  that  any  cases  of 
spontaneous  and  persistent  glycosuria  are  due  solely  to  increased 
permeability  of  the  renal  epithelium  such  as  may  be  brought  about 
experimentally  by  the  administration  of  phloridzin.  The  light  forms 
of  diabetes  resemble  alimentary  glycosuria  except  in  the  fact  that  the 
Assimilation-limit  for  carbohydrates  is  unusually  low  so  that  glycosuria 
recurs  whenever  a  normal  abundance  of  carbohydrate  is  ingested.  In 
such  cases  the  mere  performance  of  muscular  work  may  arrest  the  gly- 
cosuria. Between  this  light  form  of  diabetes  and  the  more  severe 
forms  every  intermediate  stage  may  be  observed,  and  not  infrequently 
the  same  patient  may  pass  through  all  degrees  of  severity  of  the  disease 
successively.  In  most  severe  forms  of  diabetes  sugar  continues  to  be 
eliminated  on  a  pure  protein  diet  and  the  urine  may  contain  over  ten 
per  cent,  of  glucose,  being  usually,  but  not  invariably,  dark  and  dis- 
colored from  the  presence  of  other  abnormal  constituents  arising  from 
the  disordered  metabolism. 

The  sugar  which  is  excreted  in  the  severe  forms  of  diabetes  does  not 
arise  from  carbohydrates  in  the  diet  or  in  the  tissues,  for  not  only  does 
it  continue  on  a  carbohydrate-free  diet,  but  the  quantity  excreted 
per  diem  may  be  far  in  excess  of  the  carbohydrates  in  the  food  and  in 
the  tissues  of  the  body  added  together.  Thus  in  one  experiment  upon 
a  depancreatized  dog  Pfliiger  found  that  out  of  a  total  excretion  of 
3097  grams  of  sugar  only  422  grams  could  possibly  be  accounted 
for  as  arising  from  carbohydrate  reserves  of  the  animal.  The  dif- 
ference, namely  2675  grams,  must  have  arisen  from  some  other 
source.  Liithje  even  went  so  far  as  to  feed  a  depancreatized  dog  com- 
pletely upon  casein.  In  eight  weeks  it  excreted  nearly  1200  grams 
of  sugar,  only  a  small  proportion  of  which,  of  course*  could  have 
been  derived  from  glycogen  in  the  tissues  of  the  animal.  Since  the 
fats,  upon  a  diet  such  as  this,  are  very  quickly  used  up,  we  have  no 
alternative  but  to  assume  that  the  sugar  was  derived  in  part  from  the 
decomposition  of  proteins  and,  as  a  matter  of  fact,  in  the  severer 
forms  of  diabetes  there  is  a  decided  tendency  for  the  ratio  of  the  sugar 


INTERMEDIATE  METABOLISM  OF  THE  FATS 


403 


(dextrose)  to  the  nitrogen  eliminated  in  the  diet  to  approach  a  constant 
level.  This  ratio,  designated  usually  by  the  symbol  ^  is  regarded  by 
Lusk  as  affording  valuable  indication  of  the  severity  of  the  diabetes, 
for  he  finds  that  upon  an  exclusively  fat-and-protein  diet  the  ^  ratio 
in  the  severest  cases  of  diabetes  approaches  a  critical  value  of  3.65  :  1. 
If  the  sugar  excreted  were  wholly  derived  from  protein  this  would 
mean  that  from  6.25  grams  of  protein  decomposed  in  the  tissues  of 
the  diabetic,  3.65  grams,  or  58  per  cent,  of  the  weight  of  the  protein, 
was  transformed  into  glucose.  This  Lusk  believes  to  be  the  maximal 
quantity  of  carbohydrate  which  is  obtainable  from  protein,  and  he 
illustrates  this  by  reference  to  the  following  figures: 

Maximum  —  ratios  observed  in: 


Phloridzin  diabetes. 

Diabetes  mellitus  in  man. 

In  dog. 
Lusk. 

In  man. 
Benedict. 

Mandel 
and  Lusk. 

Grunwald. 

Foster. 

Mosenthal. 

Joslin. 

3.65 
3.66 
3.62 

3.64 

3.58 
3.82 
3.66 

3.68 

3.60 
3.65 
3.66 

3.75 
3.56 
3.70 

3.64 

3.58 
3.38 

3.48 

3.75 
3.85 
3.  44 

3.66 

3.69 
3.67 
3.67 

3.68 

3.64 

According  to  Joslin  these  high  ratios,  which  usually  only  precede 
death  by  a  brief  interval,  are  never  observed  if  Fats  be  excluded 
from  the  diet,  a  fact  which  is  a  very  striking  illustration  of  the  deter- 
minative part  played  by  fats  in  the  evolution  of  diabetic  symptoms,  a 
part,  however,  which  has  only  in  recent  years  come  to  be  fully  appre- 
ciated, thanks  to  the  work  of  Allen,  Joslin,  Bloor  and  other  investi- 
gators. 

The  excretion  of  sugar  in  diabetes  mellitus  is  not  attributable  to 
loss  of  glycogen  storage-capacity  on  the  part  of  the  liver,  for  eve  a  in 
fatal  cases,  and  after  a  prolonged  excretion  of  sugar,  appreciable 
quantities  of  glycogen  may  still  be  found  in  the  liver.  It  is  naturally 
a  difficult  matter  to  ascertain  whether  or  not  the  storage  capacity  of 
the  liver  in  diabetics  is  fully  normal,  but  there  can  be  no  question 
but  that  the  main  abnormality  of  the  carbohydrate  metabolism  in 
diabetes  is  essentially  a  failure  to  utilize  the  glucose  in  the  diet.  The 
tissues  which  are  unable  to  utilize  glucose  are  nevertheless  starving 
for  it  and  every  possible  mechanism  for  manufacturing  glucose  from 
other  foodstuffs,  even  as  we  have  seen,  from  proteins,  is  pressed  into 
service,  but  the  product  of  these  efforts  is  still  glucose  and,  therefore, 
worthless  or  even  worse,  for  it  is  excreted  from  the  body  and  involves 
a  corresponding  wastage  of  the  fuel-value  of  the  dietary. 

The  failure  of  the  diabetic  to  oxidize  glucose  does  not  by  any  means 
originate  in  a  failure  of  oxidative  powers  in  general.  On  the  contrary 
the  relationship  of  the  condition  to  glucose  and  also  as  we  shall  see,  to 


404      PROCESSES  INFERRED  FROM  DIRECT  OBSERVATION 

the  fats  is  highly  specific  and  the  oxidation  of  other  and  even  much 
more  difficultly  oxidizable  substances  may  be  normal;  thus  Lactic  Acid, 
Mannitol  and  even  Inosite  or  Benzene  are  oxidized  just  as  well  by  the 
diabetic  as  by  the  normal  individual.  Even  a  very  slight  degree  of 
oxidation  of  glucose  itself  suffices  to  enable  the  tissues  to  overcome  the 
obstacle.  Thus  gluconic  acid,  glucuronic  acid,  saccharic  acid  and  mucic 
acid  are  all  readily  oxidized  by  a  diabetic.  The  relationship  of  these 
substances  to  glucos*e  may  be  seen  from  the  following  formulae 

CHO         COOH        CHO         COOH        COOH 

ill!! 

HCOH        HCOH        HCOH       HCOH       HCOH 

I  '       I         I        !        I 

HOCH        HOCH        HOCH       HOCH       HOCH 

I  I  I  I  I 

HCOH        HCOH        HCOH       HCOH      HOCH 

I  I  I  I  I 

HCOH        HCOH        HCOH       HCOH       HCOH 

r          i  i  i  i 

CH2OH  CH2OH  COOH  COOH  COOH 

Glucose.  Gluconic  acid.  Glucuronic  Saccharic  Mucic  acid. 

acid.  acid. 

Even  more  surprising  is  the  fact  that  sugars  other  than  glucose  may 
be  very  much  better  utilized  by  a  diabetic  than  glucose  itself.  Cane- 
sugar  is  badly  tolerated,  as  might  be  expected  from  the  fact  that  it 
yields  glucose  on  hydrolysis.  For  the  same  reason  Maltose,  which 
yields  two  molecules  of  glucose  when  hydrolyzed,  is  even  less  well 
tolerated  by  diabetics  than  cane-sugar.  Lactose  is  very  badly  tolerated 
probably  because  it  gives  rise,  on  hydrolysis,  not  only  to  glucose  but 
also  to  Galactose  which  is  very  poorly  assimilated  by  diabetics.  Levu- 
lose,  on  the  contrary  is  comparatively  well  assimilated.  In  many 
cases  it  is  possible  to  administer  levulose  to  diabetics  without  untoward 
symptoms  when  similar  quantities  of  glucose  would  precipitate  a  pro- 
found glycosuria.  Depancreatized  dogs  will  store  up  glycogen  on  a 
levulose  diet  when  they  cannot  do  so  on  a  diet  containing  equal  quanti- 
ties of  glucose.  For  this  reason,  since  levulose  is  somewhat  expensive, 
it  has  been  proposed  to  administer  Inulin  to  diabetics.  Inulin  is  a 
polysaccharide  of  levulose  which  occurs  in  the  tubers  of  dahlias,  the 
tuberous  artichoke  and  the  sweet  potato.  It  is,  however,  indigestible 
by  any  of  the  alimentary  juices  and  simply  increases  the  bulk  of  the 
feces  and  provides  a  culture-medium  for  intestinal  bacteria.  The 
bacteria  in  the  lower  intestine  certainly  attack  inulin  and  the  products 
of  their  activity  may  be  absorbed  or  utilized,  but  these  products  are 
not  of  a  carbohydrate  nature,  for  if  inulin  be  administered  to  an  animal 
with  phloridzin  glycosuria,  no  increase  of  sugar-output  is  observed. 
Inulin  is  therefore  of  little  if  any  value  to  a  diabetic. 

Now  it  is  a  very  significant  fact  that  when  levulose  is  tolerated  by  a 
depancreatized  animal,  it  is  converted  into  glycogen  in  the  liver. 
This  would  point,  seemingly,  to  a  failure  of  the  liver  to  convert  glucose 
into  glycogen  in  diabetics,  although  it  is  well  able  to  store  the  glycogen 


INTERMEDIATE   METABOLISM  OF   THE   FATS  405 

when  it  has  once  been  formed.  A  slight  change  in  the  configuration  of 
the  molecule  of  sugar  which  is  absorbed  and  carried  to  the  liver  enables 
the  liver  to  perform  its  customary  function. 

The  Urine  of  diabetics  has  very  frequently  a  pronounced  fruity  odor, 
and  is  usually  decidedly  acid  in  reaction.  These  characteristics  of 
diabetic  urine  are  due  to  the  presence  therein  of  extraordinary  amounts 
of  Aceto-acetic  Acid,  CH3COCH2COOH,  Acetone,  CH3COCH3  and 
Hydroxybutyric  Acid,  CH3CH(OH)CH2COOH.  These  products  are 
all  closely  related  to  one  another  and  unquestionably  arise  from  the 
same  source.  Thus  aceto-acetic  acid  may  be  derived  from  hydroxy- 
butyric  acid  by  oxidation,  water  being  split  off,  while  aceto-acetic  acid, 
with  the  loss  of  carbon  dioxide,  is  convertible  into  acetone.  It  is 
probable  that  hydroxy butyric  acid  is  the  parent  substance  of  all  the 
"acetone-bodies"  which  are  found  in  the  urine  of  diabetics.  The 
production  of  these  substances  rapidly  and  in  large  amounts,  produces 
the  extreme  Acidosis  which  is  characteristic  of  the  later  stages  of 
untreated  or  improperly  treated  diabetes,  and  which  culminates  in  the 
Diabetic  Coma  or  acid-intoxication  which  formerly  was  the  invariable 
and  still  is  the  very  frequent  termination  of  the  disease. 

The  amount  of  acetone  in  diabetic  urine  is  comparatively  small  and 
it  is  of  minor  significance.  The  aceto-acetic  acid  may  be  detected  by 
the  deep  red  color  which  is  communicated  to  urine  containing  this 
substance  if  Ferric  Chloride  solution  be  added  to  it  in  excess  of  the 
amount  necessary  to  precipitate  the  phosphoric  acid  as  ferric  phosphate. 
It  was  formerly  believed  that  the  acetone  bodies  in  urine  were  derived 
from  the  imperfect  oxidation  of  Carbohydrates  and  that  they  probably 
represented  intermediate  stages  in  the  degradation  of  carbohydrates 
to  carbon  dioxide  and  water.  This  view  has  now  been  abandoned  with 
the  recognition  of  the  fact  that  Fats  play  a  predominant  part  in  the 
genesis  of  diabetic  Acidosis.  The  very  slight  change  in  the  glucose 
molecule  which  suffices  to  render  it  assimilable  and  utilizable  points,  in 
any  case,  to  the  improbability  that  succeeding  stages  in  the  oxidation 
of  glucose  are  exceptionally  delayed  in  the  tissues  of  the  diabetic.  If 
oxybutyric  acid  were  in  truth  an  intermediate  stage  in  the  oxidation  of 
carbohydrates,  as  lactic  acid,  for  example  is  known  to  be,  then  the 
accumulation  of  this  substance  in  the  blood  and  in  the  tissues  must 
mean  that  the  subsequent  steps  of  oxidation  have  become  exceptionally 
difficult.  But  the  very  slightest  initial  oxidation  of  glucose  renders  it 
readily  utilizable,  so  that  we  must  infer  that  all  stages  of  oxidation 
succeeding  the  formation  of  gluconic  or  glucuronic  acids,  for  example, 
are  readily  performed  by  the  diabetic.  Now  oxybutyric  acid,  if  it  were 
formed  at  all  from  glucose,  must  succeed  the  formation  of  gluconic  or 
glucuronic  acids,  so  that  the  accumulation  of  this  substance  in  the 
tissues  of  a  diabetic  evidently  cannot  be  due  to  the  arrested  oxidation 
of  carbohydrates.  As  a  matter  of  fact,  Macleod  and  Pearce  have  found 
that  the  oxidation  of  glucose  in  the  tissues  of  depancreatized  or  even  in 
eviscerated  animals  is  in  no  way  defective,  and  Meltzer  and  Kleiner 


406      PROCESSES  INFERRED  FROM  DIRECT  OBSERVATION 


have  shown  that  a  large  part  of  the  glucose  which  circulates  in  the 
blood  of  a  diabetic  is  actually  utilized  by  his  tissues. 

The  examination  of  the  blood  in  diabetics  very  frequently  reveals, 
not  only  glucohemia,  but  also  a  pronounced  Lipemia,  which  may  be  so 
severe  as  to  give  to  the  centrifuged  blood-serum  a  distinctly  milky 
appearance.  The  following  are  results  obtained  by  Bloor  in  estimating 
the  lipoids  in  normal  and  in  diabetic  blood : 


Total  fatty  acids, 
grams  per  100  c.c. 

Lecithin, 
grams  per  100  c.c. 

Cholesterol, 
grams  per  100  c.c. 

Whole 
blood. 

Plasma. 

Cor- 
puscles. 

Whole 
blood. 

Plasma. 

Cor- 
puscles. 

Whole 
blood. 

Plasma. 

Cor- 
puscles. 

Diabetic  extremes 
Diabetic  average 
(34  analyses) 
Normal  average 
(19  analyses) 
Normal  extremes 

.41-.  76 
.52 

.37 
.29-.  42 

.46-.  93 
.59 

.39 
.30-.  47 

.33-.  62 
.43 

.34 
.27-.  45 

.26-.  50 
.36 

.30 
.28-.  33 

.17-.  48 
.30 

.21 
.17-.  26 

.32-.  60 
.46 

.42 
.35-.  48 

.19-.  44 
.29 

.22 
.19-.  25 

.16-.  65 
.36 

.23 
.19-.  31 

.17-.  24 
.20 

.20 
.17-.  24 

It  will  be  observed  that  the  percentage  of  all  the  lipoidal  constituents 
of  the  plasma  is  much  increased  in  diabetics,  while  the  lipoidal  con- 
stituents of  the  corpuscles  remain  comparatively  unaffected.  The 
increase  is  especially  marked  in  the  Neutral  Fats  (estimated  as  fatty 
acids)  and  in  the  Cholesterol  fractions.  The  lecithin  or  Phospholipin 
fraction  increases  also  but  in  much  less  proportion  than  the  others,  so 

.  fatty  acid        cholesterol 

that  the  ratios  — : — r-p —  or  — = — T-T-. —      are    abnormally    high    in 
lecithin  lecithin 

diabetic  blood-plasma.  For  this  reason  it  has  been  suggested  that  part 
at  least  of  the  failure  of  diabetics  to  utilize  fat  is  due  to  an  inability 
to  convert  neutral  fatty  acids  into  phospholipins. 

The  attention  of  earlier  investigators  of  diabetes  was  focussed  upon 
the  intolerance  of  these  patients  for  carbohydrates,  and  the  main 
objective  of  the  physician  was  to  decrease  the  output  of  glucose  in  the 
urine.  Carbohydrates  were  therefore  necessarily  excluded  from  the 
diet,  and  to  replace  the  deficient  calorific  value  thus  entailed  the  fats 
in  the  diet  were  not  unusually  increased.  This  procedure  frequently 
had  the  gratifying  result,  for  the  time  being,  of  reducing  or  even  elimi- 
nating the  output  of  glucose  in  the  urine,  but  sooner  or  later  the  patient, 
whose  condition  at  first  seemed  much  improved,  would  again  begin  to 
excrete  glucose;  a  severe  acidosis  developed  and  the  case  became  hope- 
less, terminating  in  diabetic  coma. 

This  result  has  been  duplicated  by  F.  M.  Allen  in  partially  depan- 
creatized  dogs,  and  he  attributes  it  to  the  progressive  degeneration  of 
the  Islets  of  Langerhans  in  the  residual  tissue  due  to  overstrain.  As  a 
source  of  protein  he  administered  beef-lung  to  the  animals,  and  suet 
was  employed  as  a  means  of  administering  fats.  The  following  is  his 
description  of  a  typical  result : 

"We  may  take  the  customary  treatment  of  moderate  diabetes  and 


INTERMEDIATE  METABOLISM  OF  THE  FATS  407 

illustrate  it  in  dogs.  Suppose  that  suitable  operation  and  overfeeding 
have  produced  a  condition  where  there  is  marked  glycosuria  on  a  kilo- 
gram of  lung,  but  sugar-freedom  on  800  grams  of  lung,  together  with 
a  fair  state  of  nutrition  and  entire  absence  of  ketonuria.  Now  place 
the  dog  on  600  to  800  grams  of  lung  and  100  to  200  grams  of  suet, 
according  to  the  classical  method.  There  is  no  glycosuria,  weight  is 
gained,  and  the  .condition  is  splendid  for  weeks  and  possibly  months. 
The  treatment  is  highly  successful.  Closer  examination  shows  the 
presence  of  hyperglycemia  and  slight  ketonuria1  which  are  usual  in 
the  patients  of  corresponding  type.  Glycosuria  follows,  illustrating 
the  spontaneous  downward  progress  which  the  authorities  describe. 
This  is  cleared  up  by  a  few  fast-days  on  the  Naunyn  plan,  and  the  diet 
is  again  adjusted;  it  may  now  be  400  grams  of  lung  and  200  grams 
of  suet.  The  gain  in  weight  continues  as  before,  with  hyperglycemia, 
ketonuria  and  subsequent  glycosuria.  Again  the  fast  days  are  used 
and  the  protein  diminished,  so  that  the  diet  is  perhaps  200  grams  of 
lung  and  200  grams  of  suet.  The  same  cycle  is  repeated.  Now  the 
dog  is  in  splendid  condition  and  spirits,  the  coat  sleek,  the  appearance 
such  that  he  might  create  a  good  impression  out  walking  in  the  park, 
only  he  has  a  difficulty  in  remaining  sugar-free  on  even  the  protein 
minimum,  and  the  fat  may  be  pushed  higher  to  maintain  nutrition 
against  the  repeated  fast  days.  If  the  dog  has  actually  been  kept  fat, 
a  fasting  period  about  this  time  may  diminish  the  glycosuria  or  it  may 
remain  high.  The  previously  lively  and  hungry  animal  begins  to  show 
a  curious  little  mournfulness,  and  complete  repugnance  to  food.  A  day 
or  two  later,  vomiting  of  clear  mucus  begins,  and  the  dog  drinks  and 
vomits  water.  The  acetone-reaction  is  heavy;  the  ferric  chloride  may 
be  heavy  or  slight.  The  alkali-reserve  of  the  blood  falls  low,  and  the 
complete  picture  of  patients  who  go  into  fatal  acidosis  on  fasting  is 
reproduced." 

As  Joslin  has  pointed  out,  patients  with  severe  diabetes  may  struggle 
on,  contending  against  many  complications,  and  surviving  for  years  on 
an  "atrocious  diet,"  but  let  a  doctor  intervene,  eliminate  carbohydrates 
from  the  diet  and  replace  them  by  an  equicalorific  allowance  of  fat, 
and  the  patient  promptly  dies  in  diabetic  coma.  The  treatment  is 
completely  successful,  no  doubt,  in  the  sense  that  glucose  temporarily 
disappears  from  the  urine,  but  the  patient  nevertheless  dies. 

Diabetes  is,  in  fact,  a  multiple  metabolic  disorder  of  which  the  failure 
to  utilize  glucose  is  merely  one  manifestation  which  only  indirectly 
induces  the  fatal  outcome.  The  exclusion  of  carbohydrates  from  the 
diet  renders  calorific  equilibrium  and  the  maintenance  of  tissue-  and 
body-weight  impossible,  unless  fat  be  partaken  of,  not  in  usual,  but 
even  in  unusual  quantities.  The  diabetic,  however,  has  a  genuine 
inability  to  oxidize  fats,  and  intermediate  products,  of  which  the  lead- 
ing examples  are  oxybutyric  and  aceto-acetic  acids,  are  formed  and 
accumulate  in  dangerous  and  ultimately  fatal  amounts. 

1  "Acetone  bodies"  in  the  urine. 


408      PROCESSES  INFERRED  FROM  DIRECT  OBSERVATION 

In  this  dilemma  the  only  feasible  procedure  is  to  take  advantage  of 
the  long-recognized  fact  that  the  tissues  may  be  educated  by  habitude 
to  the  proper  utilization  of  carbohydrates,  but  the  slightest  overstrain 
upon  the  carbohydrate-utilizing  mechanism  produces  a  directly  con- 
trary result  and  accelerates  the  downward  course  of  the  diabetic.  This 
is  the  foundation  of  "Allen's  Paradoxical  Law,"  namely,  that  "whereas 
in  normal  individuals  the  more  sugar  is  given  the  more  is  utilized,  the 
reverse  is  true  in  diabetes."  The  treatment  suggested  by  Allen  con- 
sists essentially  in  freeing  the  urine  from  glucose  by  starvation,  bearing 
in  mind,  however,  the  fact  that  starvation  increases  acidosis  and  that 
if  the  preceding  acidosis  was  high  the  additional  acidosis  of  too  severe 
or  too  prolonged  starvation  may  precipitate  Diabetic  Coma.  The 
starvation-period  is  succeeded  by  a  period  in  which  proteins  are 
admitted  to  the  diet.  Carbohydrates  are  now  admitted,  at  first  in 
very  small,  and  then  in  gradually  increasing  amounts,  until  a  tolerance 
is  built  up.  Fats  are  admitted  last  of  all,  and  with  great  caution,  the 
allowance  never  being  a  large  one.  Patients  treated  in  this  way  cannot 
commit  dietary  indiscretions,  but  they  may  maintain  a  tolerably 
normal  and  healthy  existence  for  a  number  of  years.  Whether  the 
"expectation  of  life"  of  a  diabetic  may,  by  a  systematic  regimen  of  this 
kind,  be  rendered  equal  to  that  of  a  normal  individual  of  like  age  and 
antecedents,  cannot  as  yet  be  stated,  for  the  treatment  of  diabetes 
based  upon  a  full  realization  of  the  part  played  by  fats  in  the  genesis 
of  fatal  symptoms  has  only  recently  come  into  being,  and  statistics 
are  therefore  not  available.  Furthermore  the  number  of  psychological 
factors  which  enter  into  the  successful  treatment  of  any  chronic 
disease  must  be  carefully  borne  in  mind  in  adjudging  the  statistics 
when  they  do  become  available.  The  physician  may  know  very  well 
what  ought  to  be  done,  but  in  practice  he  may  rarely  achieve  it.  The 
fluctuating  cooperation  of  attendants,  and  the  fragmentary  attention 
of  the  busy  practitioner  to  any  individual  case;  the  thousand  personal 
details  of  means,  circumstances,  behavior,  temperament,  and  metabo- 
lism, which  render  every  individual  case  a  separate  problem  which 
differs  from  any  other,  these  factors  combine  to  detract  from  the 
success  of  any  method  of  treatment  of  a  chronic  disease-condition, 
however  theoretically  perfect  the  method  may  chance  to  be.  It  is 
probable,  indeed,  that  to  correctly  evaluate  any  method  of  treatment  of 
a  chronic  condition  we  should  look  to  the  successful  cases  rather  than 
to  the  failures.  The  ideal  means  of  attaining  success  would  be,  of  course, 
to  educate  the  patient  to  become  his  own  doctor.  Unfortunately, 
however,  many  patients  are  unteachable,  and  most  physicians  are  bad 
pedagogues. 

To  revert  to  the  questions  of  intermediate  metabolism  which  render 
the  phenomenon  of  diabetes  of  such  exceptional  interest  to  the  bio- 
logical chemist;  it  appears  very  probable  that  /3-Hydroxybutyric  Acid 
is  one  of  the  normal  intermediate  steps  in  the  oxidation  of  fats,  just 
as  lactic  acid  is  an  intermediate  step  in  the  oxidation  of  carbohydrates, 


INTERMEDIATE   METABOLISM  OF   THE  FATS  409 

and  that  in  diabetics,  through  failure  of  a  particular  tissue,  namely 
the  islets  of  Langerhans  in  the  pancreas,  the  further  stages  of  oxidation 
are  hindered,  just  as,  in  asphyxia,  the  oxidations  of  carbohydrates 
subsequent  to  the  production  of  lactic  acid  are  hindered.  The  appear- 
ance of  abnormal  quantities  of  Cholesterol  in  the  blood  of  diabetics 
suggests  the  possibility  that  the  metabolism  of  the  Hydroxyaromatic 
Derivatives  is  also  disordered  in  diabetics.  The  other  u  acetone- 
bodies"  in  diabetic  urine  are  undoubtedly  derived  from  /3-hydroxy- 
butyric  acid.  Thus,  if  the  liver  be  perfused  with  blood  containing  this 
substance,  the  blood  which  issues  from  the  liver  contains  aceto-acetic 
acid,  and  even  minced  liver  will  bring  about  the  same  transformation. 

Butyric  acid  is  converted  quantitatively  by  oxidation  into  /3-hydroxy- 
butyric  acid,  whereas  Magnus-Levy  has  pointed  out  that  100  grams 
of  Neutral  Fat  made  up  of  tristearin,  tripalmitin  and  triolein  can  yield 
a  maximum  of  only  36.2  grams  of  fl-hydroxybutyric  acid.  Hence 
cream  or  Butter  Fat,  with  its  high  content  of  butyrates,  is  a  much  more 
dangerous  source  of  "acetone  bodies"  than  mutton-fat  or  bacon-fat 
or  butter-substitutes  such  as  oleomargarine.  This  fact  is  illustrated 
very  strikingly  in  the  intolerance  which  infants  frequently  display  to 
cream  or  butter,  exhibiting  decided  symptoms  of  Acidosis  when  these 
are  administered  in  what,  for  other  children,  would  be  moderate 
amounts.  These  infants  not  infrequently  tolerate  a  higher  fat,  or  even 
olive  oil,  much  better  than  they  will  tolerate  cream  or  butter. 

The  oxidation  of  the  fats  appears  to  take  place  in  a  series  of  similar 
successive  steps,  the  point  of  attack  at  each  stage  in  the  oxidation  being 
the  ^-carbon  atom,  that  is,  the  second  carbon  atom  ia  the  hydrocarbon 
chain,  counting  from  the  carboxyl-group.  Thus  Stearic  Acid  is  con- 
verted into  Palmitic  Acid  in  the  following  way: 


410      PROCESSES  INFERRED  FROM  DIRECT  OBSERVATION 


CH3 

CH3 

CH3 

CH3 

| 

1 

1 

CH2 

CH2 

i 

CH2 

CH2 

| 

CH2 

CH2 

i 

CH2 

CH2 

| 

CH2 

CH2 

CH2 

CH2 

CH2 

CH2 

CH2 

CH2 

I 

1 

1 

CH2 

CH2 

CH2 

CH2 

1 

CH2 

CH2 

CH2 

CH2 

1 

1 

CH2 

CH2 

CH2 

CH2 

I 

1 

CH2 

CH2 

CH2 

CH2 

1 

CH2 

CH2 

CH2 

CH2 

I 

1 

CH2 

CH2 

CH2 

CH2 

1 

1 

CH2 

CH2 

CH2 

CH2 

1 

1 

CH2 

CH2 

CH2 

CH2 

1 

CH2 

CH2 

CH2 

CH2 

I 

CH2 

CH2 

CH2 

CH2 

i 

0CH2       +       0 

=       CHOH 

+      O       =       CO       + 

4O        =       COOH 

•f 

CH2 

CH2 

CH2 

2CO2 

1 

+ 

COOH 

COOH 

COOH 

H20 

H20 

Stearic  acid. 

Intermediate 

Intermediate 

Palmitic  acid, 

hydroxy-acid. 

keto-acid, 

carbon  dioxide, 

water. 

water. 

In  a  similar  manner  palmitic  acid  yields  myristic 

acid,  the  next  pro- 

duct  is  lauric  acid 

and  this  is 

followed  in  succession 

by  capric,  caprylic 

and  caproic  acids. 

This  acid 

,  on  oxidation  of  its  /5-carbon  atom  yields 

butyric  acid  : 

CH» 

CH3 

CH3 

CH3 

1 

1 

CH2 

CH2 

CH2 

CH2 

CH2 

CH2 

CH2 

CH2 

1 

1 

/SCH2       +      0 

=       CHOH 

+       O        =       CO        + 

4O        =       COOH 

aCH2 

CH2 

CH2 

2GO2 

1 

| 

+ 

COOH 

COOH 

COOH 

H20 

H2O 

Caproic  acid. 

Intermediate 
hydroxy-acid. 

Intermediate 
keto-acid, 
water. 

Butyric  acid, 
carbon  dioxide, 
water. 

INTERMEDIATE  METABOLISM  OF  THE  FATS  411 

It  is  at  the  next  succeeding  stage  of  this  process  that  trouble  origi- 
nates in  the  diabetic.  In  normal  tissues  the  intermediate  hydroxy- 
and  keto-acids  are  present  only  evanescently,  being  immediately  oxi- 
dized to  the  lower  acid,  carbon  dioxide,  and  water.  In  the  diabetic  or 
in  the  depancreatized  animal  there  is  exceptional  difficulty  in  accom- 
plishing this,  it  would  appear,  especially  when  the  stage  of  butyric 
acid  has  been  reached  and  the  result  is  that  the  partial  products  of 
butyric  acid  oxidation,  /3-hydroxybutyric  acid  and  aceto-acetic  acid 
are  permitted  to  accumulate : 

CH3  CH3  CH3 

I  I  I 

/3CH2       +       O  CHOH       +       O        =       CO       +       H2O 

I  I  I 

«CH2  CH2  CH2 

I  I  I 

COOH  COOH  COOH 

Butyric  acid.  |3-hydroxybutyric  acid.  Aceto-acetic  acid. 

If,  however,  the  number  of  carbon  atoms  in  the  original  fatty-acid 
molecule  had  chanced  to  be  uneven  instead  of  even,  the  final  product 
of  this  process  would  have  been  Propionic  Acid,  CH3CH2COOH  instead 
of  butyric  acid. 

Now  the  important  discovery  has  been  made  by  Ringer,  that  pro- 
pionic  acid  is  completely  converted  into  Glucose  in  animal  tissues,  an 
^intermediate  stage  in  the  process  being,  not  improbably,  the  formation 
of  0-Lactic  Acid: 

2CH3CH2COOH  +  O2   =  2CH2OH.CH2COOH 
Propionic  acid.  j8-lactic  acid. 

He  furthermore  finds  that  when  fatty  acids  having  an  uneven 
number  of  carbon  atoms  are  administered  they  are  similarly  trans- 
formed, in  part,  into  glucose.  It  happens,  however,  that  the  acid 
radicals  of  the  normal  tissue-fats  of  our  dietary  always  contain  even 
numbers  of  carbon  atoms.  The  fatty  acids  possessing  uneven  numbers 
of  carbon  atoms  are  comparatively  rare,  and  do  not  occur  to  any 
important  extent  in  the  fats  of  the  normal  dietary,  otherwise  their 
administration  to  diabetics  would  enable  them  to  transform  the 
residual  unoxidized  fragment  of  the  fatty  molecule  into  glucose,  which 
is  harmless,  instead  of  hydroxybutyric  acid  which  is  toxic. 

The  normal  products  of  the  complete  oxidation  of  butyric  acid 
according  to  the  above  scheme,  would  be,  successively,  Acetic  Acid, 
carbon  dioxide  and  water: 

CH3  CH3  CH3  CH3 

I  I  I  I 

/3CH2     +     O      =     CHOH     +     O      =     CO     +     4O      =     COOH 

I  I  I 

«CH2  CH2  CH2  2CO2 

I  I  I  -  4- 

COOH  COOH  COOH  H2O 

+H2O 

Butyric  acid.         /3-hydroxybutyric  acid.         Aceto-acetic  acid,         Acetic  acid,  carbon 

water.  dioxide,  water. 


412      PROCESSES  INFERRED  FROM  DIRECT  OBSERVATION 

In  accordance  with  this  view,  Knoop  has  found  that  if  aromatic 
derivatives  of  fatty  acids  containing  an  even  number  of  carbon  atoms 
be  administered  to  animals,  Phenyl-acetic  Acid  appears  in  the  urine, 
while  if  aromatic  derivatives  of  fatty  acids  containing  an  odd  number  of 
carbon  atoms  in  the  molecule  be  administered,  the  phenyl-group  is 
split  off  as  Benzole  Acid  which,  as  usual,  combines  with  glycocoll  in  the 
tissues  and  appears  in  the  urine  as  Hippuric  Acid. 

OXIDIZING  ENZYMES. 

In  a  variety  of  animals  and  plants  there  are  to  be  found  substances 
which  are  capable  of  accelerating  certain  oxidations.  These  sub- 
stances, in  the  majority  of  cases,  resemble  the  hydrolyzing  enzymes 
in  the  minute  quantities  in  which  they  are  effective,  and  in  their  insta- 
bility toward  heat.  In  other  cases  they  are  thermostabile  and  even 
resist  boiling.  The  discovery  of  the  oxidizing  enzymes  we  owe  to  the 
versatile  investigator  Schonbein  (1799-1868),  who  employed  Guaiacum 
Tincture  as  a  means  of  detecting  them.  This  substance  is  tinged  blue, 
a  coloration  due  to  oxidative  changes,  by  many  tissues  and  tissue- 
fluids  in  the  presence  of  peroxides,  such  as,  for  example,  Hydrogen 
Peroxide.  It  is  found,  however,  that  the  oxidizing  ferments  do  not  by 
any  means  act  upon  all  oxidizable  substances  equally,  on  the  contrary 
there  is  a  high  degree  of  Specificity  in  their  effects.  Thus  the  enzyme 
or  group  of  enzymes  occurring  in  the  liver  and  in  the  spleen  which  oxi- 
dizes Purines,  converting,  for  example,  Xanthin  and  Hypoxanthin  into 
Uric  Acid,  does  not  attack  alcohols,  aldehydes  or  polyphenols.  On  the 
other  hand  the  alcohol  oxidizing  ferment  or  Alcoholase  which  oxidizes 
ethyl  alcohol  to  acetic  acid,  does  not  attack  purines  or  polyphenols. 

The  best-studied  examples  of  the  oxidizing  enzymes  are  those  which 
are  afforded  by  the  Laccases,  which  bring  about  the  hardening  of 
lacquer  varnish.  A  very  active  enzyme  has  been  prepared  from  the 
sap  of  Rhus  succedanea  by  Bertrand,  who  coagulates  the  sap  with 
alcohol,  redissolves  the  coagulum  in  water,  and  then  recoagulates  with 
alcohol.  The  coagulum  is  dried  in  vacno  and  is  then  obtained  as  a 
white  powder  which  is  readily  soluble  iu  water,  and  is  characterized 
by  its  high  content  of  Manganese.  The  activity  of  the  laccase  in  oxidiz- 
ing polyphenols  is,  in  fact,  dependent  upon  the  presence  of  manganese. 
Thus  Bertrand,  in  studying  the  action  of  a  similar  substance  from 
lucerne  with  and  without  the  aid  of  manganous  salts,  obtained  the 
following  results: 

Manganous  salt  alone  .      .      .      .      ...      .      .      0.3  c.c.  oxygen  absorbed 

Laccase  from  lucerne,  alone     .......      0.2    "         "  " 

Laccase  plus  manganous  salt 6.3"         "  " 

We  know  that  in  many  cases  the  oxides  of  polyvalent  metals  may  act 
as  carriers  of  oxygen,  through  the  intermediate  formation  of  Peroxides 
which  are  more  active  oxidizing  agents  than  free  oxygen  itself.  An 


OXIDIZING  ENZYMES  413 

example  which  is  very  familiar  to  biological  chemists  is  that  afforded 
by  the  action  of  alkaline  copper  salts  upon  glucose.  If  a  limited 
quantity  of  Fehling's  Solution  be  run  into  a  boiling  solution  of  glucose 
the  solution  is  decolorized  and,  the  red  cuprous  oxide  is  precipitated, 
but  upon  exposing  the  mixture  in  a  shallow  vessel  to  the  air,  the 
cuprous  oxide  again  takes  up  oxygen,  passes  into  solution  and  tinges 
the  fluid  blue.  If  the  mixture  be  now  boiled,  the  cupric  hydroxide 
again  parts  with  its  oxygen  to  the  excess  of  glucose,  so  that  if  the  process 
be  repeated  a  sufficient  number  of  times  a  limited  quantity  of  Fehling's 
solution  will  oxidize  a  relatively  unlimited  quantity  of  glucose.  This 
is,  in  fact,  the  chief  pitfall  in  the  practical  employment  of  Fehling's 
method  of  sugar  estimation.  In  a  similar  manner  many  other  metal 
oxides  are  capable  of  acting  as  activators  or  carriers  of  oxygen. 

According  to  Bach  and  Chodat  the  oxidizing  enzymes  which  occur 
in  the  majority  of  living  tissues  in  reality  consist  of  two  parts:  the 
one  part,  the  Oxygenase,  playing  the  role  that  cuprous  oxide  plays  in 
the  oxidation  of  sugar,  namely  that  of  a  carrier  of  oxygen,  while  the 
other  part,  the  Peroxidase,  facilitates  the  transfer  of  the  oxygen  from 
the  oxygenase  to  the  material  which  is  undergoing  oxidation.  Hydrogen 
Peroxide  may  in  many  cases  take  the  place  of  the  oxygenase,  and  hence 
we  obtain  the  blueing  of  guaiacum  tincture  when  blood  or  a  tissue- 
extract  together  with  hydrogen  peroxide  act  upon  it.  The  function  of 
the  manganous  salt  in  Laccase  appears  to  be  associated  with  the 
oxygenase  fraction,  while  in  many  oxidizing  ferments  found  in  animal 
and  other  plant  tissues,  Iron  plays  the  role  which  manganese  plays  in 
determining  the  activity  of  laccase.  In  this  connection  it  is  of  great 
interest  to  note  that  Hemoglobin  is  itself  an  oxygenase,  and  that, 
according  to  Bertrand,  the  Benzidine  and  Guaiacum  tests  for  blood  are, 
in  actuality,  tests  for  hemoglobin. 

The  majority  of  the  oxidizing  enzymes  appear  to  be  substances  of  a 
complex  character,  in  many  cases  either  protein  in  nature  or  closely 
associated  with  protein.  It  is  probable  that  the  majority  of  the 
oxygenases  or  oxygen-carriers  are  bodies  analogous  in  complexity  to 
hemoglobin  and,  like  hemoglobin,  containing  iron  or  some  other  polyv- 
alent metal  as  an  integral  portion  of  the  molecule.  On  the  other  hand 
Euler  and  Bolin  have  shown  that  the  Laccase  from  Medicago  saliva 
is  a  relatively  simple  substance,  being  a  mixture  of  the  calcium  salts  of 
aliphatic  hydroxy-acids.  A  synthetic  mixture  of  the  calcium  salts  of 
glycollic,  citric,  malic  and  mesoxalic  acids  was  found  by  them  to  exert 
the  same  action  in  accelerating  the  oxidation  of  polyphenols  as  the 
natural  laccase.  This  enzyme  is,  of  course,  thermostabile. 

An  important  group  of  oxidizing  enzymes  is  that  of  the  Tyrosinases 
which  convert  tyrosine  into  dark-colored  substances  of  complex  struc- 
ture known  as  Melanins,  which  are  probably  identical  with  or  closely 
allied  to  many  naturally-occurring  pigments.  These  enzymes  are 
found  in  many  vegetable  tissues  and  von  Furth  has  also  found  them 
in  the  tissue-fluids  of  many  insects  and  in  the  ink-sac  of  the  cephalopod 


414      PROCESSES  INFERRED  FROM  DIRECT  OBSERVATION 

Sepia.    These  oxidases  are  also  able  to  accomplish  the  oxidation  of 
other  hydroxy-aromatic  compounds,  such  as  Catechol  and  Quinol. 

An  enzyme  which  is  often  mistakenly  regarded  as  an  oxidizing 
enzyme  is  the  Catalase  which  occurs  in  nearly  all  living  tissues  and 
which  possesses  the  property  of  decomposing  peroxides,  without, 
however,  liberating  active  oxygen.  It  is  to  this  enzyme  that  the 
frothing  of  hydrogen  peroxide  when  added  to  blood  or  saliva  is  due. 
It  is  in  fact  a  retarder  of  oxidations  and  not  an  accelerator,  for  it  antici- 
pates the  action  of  peroxidase  upon  peroxides  and  decomposes  them, 
thus  depriving  them  of  ability  to  transfer  oxygen  to  oxidizable  mate- 
rials. It  is  probably  to  be  regarded  as  a  controlling  agent  or  check  upon 
overactivity  of  oxidizing  enzymes.  It  has  been  shown  by  Burge  that 
the  catalase-content  of  different  tissues  varies  very  greatly,  that  of  the 
liver  being  greatly  in  excess  of  the  catalase-content  of  muscular  tissues. 
The  curious  observation  has  been  made  by  Burnett,  however,  that  if  a 
small  proportion  of  liver-tissue  be  mixed  with  muscle-tissue  the  power 
of  this  mixture  to  decompose  hydrogen  peroxide  is  equal  to  that  of 
an  equal  weight  of  pure  liver-tissue.  This  looks  either  as  if  catalase 
really  consists,  like  the  oxidases,  of  two  parts,  of  which  only  one  is 
contained  in  considerable  amount  in  muscle-tissue,  or  else  the  catalase 
in  muscle-tissue  is  present  therein,  not  as  such,  but  in  the  form  of  a 
proenzyme  or  zymogen,  which  is  activated  by  liver-tissue. 

BIOLUMINESCENCE. 

The  phenomenon  of  bioluminescence  or  "phosphorescence"  which  is 
displayed  by  so  many  organisms,  both  vegetable  and  animal,  has 
recently  been  subjected  to  very  careful  study  and  analysis  by  N. 
Harvey.  The  peculiarity  of  bioluminescence  is  the  extraordinary 
intensity  of  the  light  which  is  developed,  without  any  perceptible 
waste  of  energy,  in  the  form  of  heat,  an  ideal  unattainable  by  any 
means  of  illumination  at  present  within  our  control.  The  luminescence 
is  dependent  upon  the  occurrence  of  oxidations,  for  it  disappears  when 
the  luminescent  system  is  deprived  of  oxygen,  even  when  the  lumines- 
cence is  made  to  occur  independently  of  the  life  of  the  organism,  as  in 
extracts  made  from  the  luminous  tissues. 

Both  Dubois  and  Harvey,  the  two  leading  investigators  of  this 
phenomenon,  are  agreed  that  the  production  of  luminescence  in 
animal  or  plant-tissues  or  tissue-extracts  requires  the  interaction  of 
two  substances.  The  one  of  these  is,  according  to  Harvey,  the  sub- 
stance from  which  the  luminescence  proceeds,  and  it  is  progressively 
consumed  in  the  process;  this  he  terms  the  Photogenin.  The  other 
substance  facilitates  the  oxidation  of  the  photogenin  and  is  termed  by 
Harvey  Photophelein.  The  photogenin  is  colloidal,  i.  e.,  does  not  pass 
through  a  dialyzing  membrane  of  parchment,  and  its  light-producing 
ability  is  destroyed  by  heating.  The  photogenin  from  the  luminous 
crustacean,  Cypridina,  at  all  events,  appears  to  be  a  protein.  It  is 


BIOL  VMINESCENCE  415 

associated  with  iron,  copper  and  manganese,  but  whether  the  presence 
of  these  metals  is  essential  to  its  luminescence  has  not  yet  been  ascer- 
tained. The  proteolytic  enzymes  destroy  its  light-producing  power. 
Photophelein,  on  the  other  hand,  appears  to  be  a  substance  related  to 
the  proteoses  or  peptones  in  many  of  its  properties,  but  it  is  not  digest- 
ible by  proteolytic  enzymes  and  it  is  soluble  in  alcohol. 

The  separation  of  photophelein  and  photogenin  from  one  another 
may  be  accomplished  by  extracting  the  luminescent  animals  or  organs 
with  hot  water.  This  extracts  the  photophelein  and  destroys  the  pho- 
togenin. A  solution  of  photogenin  may  be  prepared  by  extracting  a 
luminous  organ  with  cold  water  and  allowing  the  extract  to  stand  until 
all  luminescence  has  disappeared,  when  the  photophelein  has  been 
apparently  exhausted.  On  now  mixing  these  two  non-luminous  solu- 
tions a  bright  luminescence  at  once  appears. 

The  actual  source  of  light  has  been  shown  by  Harvey  to  be  the 
photogenin,  in  the  following  ingenious  manner:  The  light  emitted  by 
the  Eastern  American  firefly  Photinus  is  orange  in  color,  while  that 
emitted  by  Photuris  is  greenish-yellow.  If,  now,  photinus  photogenin 
is  mixed  with  photophelein  from  either  Photinus  or  Photuris  the  color 
of  the  luminescence  is  that  emitted  by  Photinus,  namely  orange,  and 
conversely  Photuris  photogenin  yields  greenish-yellow  light  whether 
the  source  of  photophelein  be  Photuris  or  Photinus.  Evidently, 
therefore,  the  character  of  the  light  emitted  is  determined  by  the 
photogenin  and  not  by  the  photophelein. 

The  action  of  photophelein  is,  to  a  limited  extent,  specific.  Thus 
firefly-photophelein  will  cause  emission  of  light  by  photogenin  derived 
from  other  insects,  but  none  from  photogenin  derived  from  crusta- 
ceans. On  the  other  hand  photogenin  may  be  caused  to  luminesce 
by  many  substances  which  are  not  of  animal  and  vegetable  origin,  and 
particularly  by  fat-solvents  and  other  Cytolytic  Agents.  Thus  lumi- 
nescence of  photogenin  may  be  caused  by  ether,  chloroform,  saponins 
or  bile-salts.  Harvey  believes  that  these  substances  promote  oxidation 
of  the  photogenin  by  increasing  the  fineness  of  the  subdivision  of  the 
colloidal  particles  of  which  it  is  composed,  and  thus  increasing  the 
area  of  exposure  to  oxygen. 

The  part  played  by  photogenin  itself  may  also  be  imitated  by  a 
variety  of  reagents.  Thus  many  aldehydes,  polyphenols  such  as 
pyrogallol,  terpenes,  waxes,  glucose,  lecithin,  cholesterol,  cetyl  and 
myricyl  alcohols,  tannic  and  gallic  acids,  certain  peptones  and  the  bile- 
acids  will  emit  luminescence  when  treated  in  certain  concentrations 
with  specific  oxidizing-agents.  Pyrogallol,  for  example,  will  luminesce 
when  treated  with  plant  Peroxidases  or  with  Hemoglobin  or  by  certain 
salts  such  as  potassium  permanganate  and  potassium  ferrocyanide, 
if  hydrogen  peroxide  is  also  present.  For  each  oxidizer  and  oxidizable 
substance  there  is  an  optimal  concentration  above  and  below  which  the 
light-emission  diminishes.  Thus  one-molecular  pyrogallol  solution 
will  give  no  light  if  mixed  with  ^  potassium  ferrocyanide  and  a  little 


416      PROCESSES  INFERRED  FROM  DIRECT  OBSERVATION 

three  per  cent,  hydrogen  peroxide,  but  T^  or  T^  pyrogallol  will 
give  a  bright  light,  while  the  light  from  T^-?W75-  pyrogallol  is  only  just 
visible.  On  the  other  hand  f  potassium  ferrocyanide  gives  no  light 
with  a  mixture  of  -nnr  pyrogallol  and  hydrogen  peroxide,  while  f^- 
potassium  ferrocyanide  causes  bright  light-emission. 

The  phenomenon  of  bioluminescence  therefore  depends  upon  the 
simultaneous  presence,  in  solution,  of  a  special  type  of  oxidizable 
substance,  and  an  oxidizing  agent  which  presents  some  analogies  to 
an  oxidase,  but  is  thermostabile  and  diffusible,  and  to  some  extent  used 
up  in  the  reaction  which  it  accelerates. 

REFERENCES. 

INTERMEDIATE  METABOLISM: 

Pfluger:     Pfluger's  Arch.,  1903,  96,  p.  1. 

Van  Hoogenhuyze  and  Verploegh:     Zeit.  f.  physiol.  Chemie,  1905,  46,  p.  415. 

Knoop:     Beitr.  z.  chem.  Physiol.  u.  Pathol.,  1905,  6,  p.  150. 

Von  Noorden:  Metabolism  and  Practical  Medicine,  English  edition,  edited  by 
Walker  Hall,  London,  1907. 

Shaffer:     Am.  Jour.  Physiol.,  1908,  22,  p.  445. 

Dakin:     Oxidations  and  Reductions  in  the  Animal  Body,  London,  1912. 

Taylor:     Digestion  and  Metabolism,  Philadelphia,  1912. 

Bloor:  Jour.  Biol.  Chem.,  1912,  11,  pp.  141  and  429;  1913,  15,  p.  105;  1913-14,  16, 
p.  517;  1914,  17,  p.  377;  1914,  19,  p.  1;  1915,  22,  p.  133;  1915,  23,  p.  317. 

Sansum  and  Woodyatt:     Ibid.,  1915,  21,  p.  1. 

Wilder:     Ibid,,  1917,  31,  p.  59. 

Hoagland  and  Mansfield:  Ibid.,  1917,  31,  p.  501.  (Glucolysis  in  Tissue,  consult 
foi  literature.) 

Lusk:     The  Science  of  Nutrition,  Philadelphia,  1919. 
PRODUCTS  OF  MUSCULAR  ACTIVITY  IN  STARVATION,  ETC  : 

Cathcart:     Jour.  Physiol  ,  1906-7,  35,  p.  500. 

Benedict:     Carnegie  Institution  of  Washington  Bull.,  1907,  77. 

Mendel  and  Rose:     Jour.  Biol.  Chem.,  1911-12,  10,  p.  213. 

Mellanby:     Proc.  Roy.  Soc.  B.,  1912-13,  86,  p.  88. 

Meyers  and  Fine:     Jour,  Biol.  Chem.,  1913,  15,  p.  305. 

Morse:     Jour.  Am.  Med.  Assn.,  1915,  65,  p.  1613. 
DIABETES: 

Allen:     Glycosuria  and  Diabetes,  Boston,  1913. 

Macleod:     Diabetes,  its  Pathological  Physiology,  1913. 

Joslin:     The  Treatment  of  Diabetes  Mellitus,  Philadelphia,  1917. 
OXIDIZING  ENZYMES: 

Bach  and  Chodat:     Centr.  f.  Biochemie,  1903,  1,  pp.  417  and  457. 

Batelli  and  Stern:  C.  Rend.  Acad.  d.  Sc.,  1905,  140,  pp.  1197  and  1352;  1905,  141, 
p.  139.  Biochem.  Zeit.,  1914,  67,  p.  443. 

Kastle:  The  Oxidases,  Hygienic  Lab.,  U.  S.  A.  Pub.  Health  and  Marine  Hospital 
Service  Bull.,  No.  59,  Washington,  1910. 

Euler:     General  Chemistry  of  the  Enzymes,  trans,  by  Pope,  New  York,  1912. 
CATALASE: 

Burge;     Am.  Jour,  Physiol.,  1916,  41,  p.  153;   1916-17,  42,  p.  600;  1917,  44,  p.  290. 

Burnett:     Proc.  Soc.  Exp.  Biol.  and  Med.,  1918,  15,  p.  80. 
BIOLUMINESCENCE  : 

Dubois:     La  Vie  et  la  Lumiere,  Paris,  1914.  4 

Harvey:  Science,  N.  S.,  1916,  44,  p.  652;  1917,  46,  p.  241.  Am.  Jour.  Physiol., 
1916,  41,  p.  454;  1916-17,  42,  p.  318.  Jour.  Biol.  Chem.,  1917,  31,  p.  311.  Jour. 
Gen.  Physiol.,  1918,  1,  pp.  133  and  269. 


CHAPTER  XVIIt. 

PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION; 

THE  ENERGY-TRANSFORMATIONS  IN 

LIVING  ORGANISMS. 

THE  INFLUENCE  OF  TEMPERATURE  UPON  LIFE-PROCESSES. 

The  influence  of  elevation  of  temperature  upon  a  chemical  reaction 
may  be  twofold.  If  the  reaction  is  at  all  exo-  or  endothermic,  that  is, 
if  any  heat  is  liberated  or  absorbed  during  the  progress  of  the  reaction, 
an  elevation  of  temperature  will  bring  about  a  definite  change  in 
Equilibrium  so  that  at  the  conclusion  of  the  reaction  the  final  relative 
proportion  of  the  various  components  is  altered.  On  the  other  hand 
a  rise  in  temperature  always  accelerates  the  attainment  of  equilibrium 
whatever  the  station  of  equilibrium  may  chance  to  be.  Thus,  not- 
withstanding the  fact  that  the  majority  of  the  hydrolyses  which  occur 
in  living  tissues  are  exothermic,  so  that  a  rise  in  temperature  tends  to 
shift  the  equilibrium  in  the  direction  of  less  complete  hydrolysis,  yet 
the  rate  of  Hydrolysis  being  more  than  proportionately  accelerated, 
enzymatic  hydrolyses  which  are  barely  perceptible  at  low  tempera- 
tures become  extremely  rapid  at  the  body-temperature  of  warm- 
blooded animals. 

The  effect  of  temperature  upon  the  velocity  of  a  chemical  reaction 
may  be  expressed  by  the  equation  : 

,  /Ti-To\ 


in  which  "ki,"  and  "k0"  signify  the  velocity-constant  at  the  absolute 
temperatures  "Ti"  and  "T0"  respectively,  "e"  is  the  base  of  the 
Napierian  logarithms  and  ^  is  a  constant,  differing  in  different  reactions, 

but  almost  invariably  possessed  of  such  a  value  that  the  ratio  ^ 

ko 

exceeds  2  when  TI  —  T0  =  10°.  The  Temperature-coefficient  of  a  chemical 
reaction  therefore,  or  the  ratio  : 

Velocity  of  the  reaction  at  T  +  10° 
Velocity  of  the  reaction  at  T 

is  in  almost  every  case  greater  than  2  and  may  be  very  greatly  in 
excess  of  this  value. 

27 


418    PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

The  behavior  of  physical,  that  is  to  say  molecular  phenomena  rather 
than  atomic,  which  are  affected  by  temperature,  is  quite  different. 
The  effect  of  temperature  is  in  these  phenomena  quantitatively  much 
less  than  it  is  in  phenomena  which  arise  from  chemical  transforma- 
tions. Thus  the  Viscosity  of  a  liquid  is  diminished  by  an  elevation  of 
temperature,  it  is  true,  but  the  reduction  of  viscosity  which  is  brought 
about  by  a  rise  of  ten  degrees  in  temperature  does  not  exceed  about 
twenty  per  cent.,  so  that  the  ratio: 

Viscosity  at  T  +.10° 


Viscosity  at  T 

is  1.2  or  thereabouts.  Consequently  all  the  physical  phenomena  in 
solutions  which  are  dependent  upon  the  viscosity  of  the  solvent,  such  as 
electrical  Conductivity,  and  Diffusion  are  affected  in  a  similar  degree 
by  elevation  of  temperature.  Those  phenomena  of  which  the  rate  is 
determined  by  changes  of  Surface-tension  have,  in  fact,  a  temperature- 
coefficient  of  less  than  unity,  the  velocity  of  changes  in  capillary 
tension  being  actually  reduced  by  elevation  of  temperature. 

One  consequence  of  this  decided  quantitative  difference  between 
the  effects  of  temperature  upon  chemical  and  physical  phenomena  is 
that  we  may,  with  a  fair  degree  of  confidence,  employ  the  temperature- 
coefficient  of  a  complex  phenomenon  which  involves  physical  as  well  as 
chemical  changes  as  a  means  of  gauging  the  extent  to  which  the  velocity 
of  the  process  is  governed  by  the  chemical  transformations  which  it 
involves.  If  the  pace  is  set  by  the  rate  at  which  some  chemical  change 
transpires,  then  the  rapidity  of  the  process  will  be  at  least  doubled 
and  not  improbably  more  than  doubled  by  a  rise  of  ten  degrees  in 
temperature.  But  if  the  chemical  transformations  are  subordinate  to 
some  physical  process  and  must  await  its  development  before  they  can 
proceed,  or  if  they  are  simply  consequent  upon  physical  changes  such 
as  electrolysis,  or  alterations  in  surface-tension,  then  the  pace  of  the 
whole  process  will  be  set  by  this  physical  event  and  the  temperature- 
coefficient  of  the  process  may  be  expected  to  be  less  than  2  or  even  very 
considerably  less  than  2. 

We  have  already  seen  that  the  various  enzymatic  hydrolyses  which 
occur  in  the  digestion  of  the  foodstuffs  yield  temperature-coefficients 
which  lie  between  2  and  4;  all  of  them  exceeding  2  at  temperatures 
which  are  not  too  far  above  the  temperature  of  the  warm-blooded 
animals.  The  temperature-coefficient  of  enzymatic  processes  neces- 
sarily declines  very  rapidly  at  temperatures  which  are  much  in  excess 
of  40°,  because  at  these  temperatures  the  acceleration  of  the  auto- 
destruction  of  the  Enzyme  itself  is  so  great  that  its  loss  of  activity  more 
than  compensates  for  the  gain  in  the  velocity  of  the  hydrolysis  which 
the  residual  undestroyed  enzyme  is  able  to  bring  about.  We  have,  in 
fact,  to  deal  with  the  resultant  of  two  opposed  processes  both  of  which 
are  accelerated  by  elevation  of  temperature.  At  lower  temperatures 


INFLUENCE  OF  TEMPERATURE   UPON  LIFE-PROCESSES     419 

the  acceleration  of  hydrolysis  is  the  predominant  result  of  raising  the 
temperature,  but  at  higher  temperatures,  destruction  of  the  enzyme 
becomes  the  controlling  factor.  The  temperature-coefficient  for 
enzyme  destruction  is  exceptionally  high,  so  that  the  rate  of  auto- 
destruction  may  be  imperceptible  between  30°  and  40°  and  extremely 
rapid  at  temperatures  lying  between  40°  and  50°. 

Even  in  a  single  uncomplicated  chemical  transformation  the  tem- 
perature-coefficient is  not  constant,  for,  reverting  to  the  equation: 


ko 

we  see  that  the  temperature-coefficient  for  10°  temperature-interval 
is  given  by: 

i  n(-M-\      ' 

KI     =     e^VTi    T0) 

k0 

it  is  therefore  not  independent  of  the  absolute  magnitude  of  the  tem- 
perature employed;  in  fact  the  temperature-coefficient  must  invariably 
decrease  as  the  temperature  rises.  Assuming  a  value  of  /z  (  =  13,  200) 
which  would  yield  a  coefficient  of  2  between  the  temperatures  of  30° 
and  40°,  the  following  table  shows  the  coefficients  which  might  be 
anticipated  at  other  temperatures  : 

Temperature- 

Temperature  interval.  coefficient. 

0°to  10°  .................      2.34 

10°  to  20°  .................      2.22 

20°  to  30°  .................      2.11 

30°  to  40°  .................      2.00 

40°  to  50°  .      .      ...      ......      ......      1-92 

The  reduction  of  the  coefficient  for  enzyme  reactions  at  temperatures 
above  40°  is,  however,  much  more  extreme  than  could  be  accounted  for 
in  this  fashion,  the  coefficient  ultimately  falling  to  zero  at  the  thermal 
limit  for  the  activity  of  the  enzyme. 

The  actual  phenomena  of  life  are  almost  invariably  of  a  mixed 
character,  involving  physical  as  well  as  chemical  processes  and  changes, 
and  we  may  inquire  through  the  investigation  of  their  temperature- 
coefficients  whether  the  physical  or  the  chemical  factors  predominate 
in  determining  the  rate  of  performance;  whether  the  chemical  trans- 
formations, in  other  words,  are  consequent  upon  preceding  physical 
changes  or  whether,  on  the  contrary,  the  physical  modifications  of 
protoplasm  await  and  are  the  resultant  of  the  chemical  transformations 
which  accompany  the  performance  of  vital  activities. 

The  first  investigator  to  apply  this  criterion  to  the  study  of  life- 
phenomena  was  Cohen,  who  in  1892  pointed  out  that  the  previous 


420    PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

measurements  by  Clausen  of  the  rate  of  production  of  Carbon  Dioxide  by 
germinating  seeds  showed  that  this  process  is  approximately  doubled 
in  velocity  by  an  elevation  of  10°  in  temperature  until  an  upper  limit 
somewhat  exceeding  40°  is  attained,  when  the  rate  of  the  tissue-respira- 
tion falls  off  owing  to  heat-injury.  This  method  of  inquiry  was  extended 
to  animal  tissues  by  C.  D.  Snyder,  who  investigated  the  influence  of 
temperature  upon  the  Rate  of  the  Heart-beat  in  the  isolated  heart  of 
the  Pacific  terrapin,  Clemmys  marmorata.  The  following  are  illus- 
trative results : 


Number  of  heart-beats  per  minute. 

Time  of  exposure 
to  the  temperature 

Heart  1. 

Heart  2. 

Heart  3. 

Heart  4. 

Heart  5. 

Heart  6. 

minutes. 

T.  =  10°. 

T.  =  20°. 

T.  =  30°. 

5 

9.5 

9.5 

21.5 

21.0 

48 

48 

10 

7.0 

9.0 

21.0 

24.0 

48 

44 

15 

6.7 

8.7 

19.0 

18.0 

48 

40 

20 

7.0 

8.2 

19.0 

16.5 

41 

30 

7.0 

7.0 

16.0 

14.0 

40 

6.5 

7.9 

15.5 

15.5 

50 

6.5 

7.9 

13.5 

16.0 

60 

6.2 

7.4 

13.0 

15.0 

It  is  evident  that  the  rate  of  the  beat  is  approximately  doubled  for 
each  10°  rise  in  temperature.  From  the  data  quoted  and  others 
obtained  by  Snyder  the  following  average  coefficients  may  be  com- 
puted: 

HEART-BEAT  OF  CLEMMYS  MARMORATA 

Temperature-coefficient 
Temperature-interval.  for  10°  intervals. 

10°  to  20° 2.3 

20°  to  30° 22 

30°  to  37° '.      ...      1.6 

at  temperatures  exceeding  37°  the  rate  of  the  beat,  instead  of  increas- 
ing, diminished  until  the  heart  came  to  a  standstill  owing  to  irreparable 
heat-injury.  These  experiments  were  subsequently  repeated  upon 
the  isolated  heart  of  another  species  of  terrapin,  Emys  europea,  by 
Galeotti  and  Piccinini,  and  by  Snyder  upon  the  isolated  heart  of  the 
frog,  and  by  Kanitz  upon  the  isolated  mammalian  heart. 

The  heart  in  situ  is,  however,  considerably  modified  in  its  behavior 
and  particularly  in  the  rate  of  beat  by  the  nervous  control  to  which  it 
is  subjected.  The  study  of  the  heart-beat  in  the  intact  animal  there- 
fore involves  more  numerous  and  more  complex  factors  than  that  of  the 
beat  of  the  excised  heart.  Nevertheless  in  this  case  also  the  rate  of 
the  beat  is  primarily  determined  by  the  velocity  of  underlying  chemical 
changes.  Thus  in  the  minute  transparent  fresh-water  crustacean, 
Ceriodaphnia,  the  heart  can  be  viewed  through  the  body-wall  of  the 


INFLUENCE  OF  TEMPERATURE   UPON  LIFE-PROCESSES     421 

animal  and  the  beats  counted  at  a  variety  of  temperatures.  The 
following  are  illustrative  of  the  results  obtained  by  this  method : 

Temperature  interval.  Temperature-coefficient. 

Il°to21° 2.76 

15°  to  25° 2.24 

17°  to  27° 2.05 

19°  to  29° 2.06 

21°  to  31° 1.14 

at  a  temperature  slightly  above  31°  the  heart-beat  ceases  and  the 
organism  dies. 

In  the  case  of  the  crustacean  Limulus  the  Heart-ganglion  can  be 
heated  or  subjected  to  other  manipulation  without  directly  involving 
th,e  heart-muscle  itself,  and  Carlson  has  found  that  by  heating  the 
ganglion  alone  the  heart-beat  is  accelerated,  the  unusually  high  tem- 
perature-coefficient of  4  being  obtained. 

On  the  other  hand,  in  the  Embryonic  Heart,  in  which  the  mechanism 
of  nervous  control  is  probably  not  yet  established,  the  rate  of  the 
heart-beat  is  similarly  affected  by  temperature,  being  doubled  or  trebled 
by  a  rise  of  ten  degrees.  The  following  are  results  obtained  by  Loeb 
and  Ewald,  employing  the  embryos,  still  enclosed  within  the  egg,  of 
the  marine  fish  Fundulus: 

Time  required  for  19 

heart-beats  in  the 
Temperature.  embryo;  seconds. 

30°  .   .   '.' 6.25 

25° 8.5 

20°  ./.,,,; 11.5 

15°  . 19.0 

10° 32.5 

5° 61.0 

10° 33.5 

15°  .   ;_ 18.8 

20° 12.0 

25°  .  '. 10.0 

30°  ....  .   .   .   .  , 6.0 

It  is  evident,  therefore,  that  both  the  muscular  and  nervous  mechan- 
isms involved  in  the  regulation  of  the  rate  of  the  heart-beat  are  pri- 
marily conditioned,  as  to  their  velocity,  by  underlying  chemical 
transformations. 

Loeb  and  Ewald  have  drawn  attention  to  the  fact  that  in  Fundulus 
embryos  the  rate  of  the  heart-beat  is  almost  the  same  in  all  the  embryos 
exposed  to  the  same  temperature,  provided  they  still  remain  enclosed 
within  the  egg.  This  is  because  of  the  elimination  of  all  secondary 
disturbing  factors.  As  soon  as  the  embryos  begin  to  move,  this 
equality  disappears,  because  the  motility  of  different  embryos  differs 
and  the  products  discharged  from  the  contracting  muscles  influence 
the  rate  of  the  heart-beat.  In  man  and  in  other  higher  animals,  the 
number  of  the  disturbing  factors,  while  the  heart  remains  in  situ,  are 
so  great  that  no  uniformity  of  rate  at  any  given  temperature  can  be 
expected.  "  Differences  in  emotions  or  the  internal  secretions  following 


422  PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

the  emotions,  differences  in  metabolism,  differences  in  the  use  of  nar- 
cotics or  drugs,  and  differences  in  activity  are  only  some  of  the  number 
of  variables  which  enter"  (Loeb).  Hence  the  attempt  to  compute  the 
temperature-coefficient  of  the  heart-beat  in  situ  in  man  from  the 
acceleration  of  the  beat  in  Fevers  is  illogical,  and  we  find,  as  a  matter  of 
fact,  a  great  deal  of  discordancy  in  the  values  computed  from  data  of 
this  kind,  coefficients  varying  from  1.8  to  5  having  been  reported. 

The  Respiratory  Rhythm  is  even  more  susceptible  to  modification  by 
sensory  stimuli,  muscular  exertion  and  so  forth,  than  the  cardiac 
rhythm,  and  consequently  the  coefficients  which  have  been  observed 
for  the  rate  of  the  respiratory  movement  at  different  temperatures  are 
not  of  so  uniform  a  character  as  those  which  are  cited  above.  Never- 
theless the  influence  of  temperature  upon  the  Respiratory  Center  is 
extremely  striking.  It  has  long  been  a  familiar  fact  that  warming  the 
blood  in  the  carotid  artery,  by  causing  it  to  flow  through  a  heated  tube, 
results  in  a  marked  acceleration  of  the  respiratory  rhythm,  and  in 
frogs  it  has  been  shown  that  the  direct  application  of  heat  to  the  floor 
of  the  fourth  ventricle  leads  to  a  very  decided  increase  in  the  rapidity 
of  respiratory  movements.  It  is  an  extremely  interesting  fact  that  the 
effect  of  temperature  upon  the  respiratory  rhythm  of  cold-blooded 
animals  is  very  much  greater  at  a  low  oxygen-tension  than  at  a  high, 
possibly  because  when  the  oxygen-tension  is  low  and  the  consumption 
of  oxygen  by  all  of  the  tissues  is  accelerated  by  an  elevation  of  tempera- 
ture, the  effect  of  the  temperature  elevation  itself  is  aided  by  the 
stimulation  of  the  respiratory  center  which  lack  of  oxygen  indirectly 
entails,  while  when  oxygen  is  abundant  there  is  sufficient  for  the  needs 
of  all  the  tissues  even  at  high  temperatures,  and  the  secondary  stimula- 
tion of  the  center  does  not  occur. 

The  influence  of  temperature  upon  the  velocity  of  "Basal  Metab- 
olism" or  tissue-respiration  can  only  be  studied  in  cold-blooded 
animals  under  conditions  which  exclude  muscular  movement,  which 
would,  of  course,  introduce  irregular  fluctuations  in  the  rate  of  con- 
sumption of  oxygen.  This  problem  has  been  approached  by  Krogh  in 
several  ingenious  ways.  One  method  was  to  employ  the  pupae  of 
insects  in  which  tissue-respiration  is  of  course  maintained  but  muscular 
movement  is  arrested.  The  following  were  results  obtained  with  the 
pupae  of  the  mealworm,  Tenebrio  molitor: 

Oxygen-consumption  Temperature-coefficient 

lemperature.  per  kilogram-hour.  per  10°  C. 

10 43.5  5.7 

15 104.  3.2 

20 185.  2.6 

300.  2.2 

445.  2.2 


25. 
30. 

32. 5C 


529. 


It  will  be  observed  that  the  temperature-coefficient  is  very  high  at 
low  temperatures  and  falls  rapidly  as  the  temperature  rises.     A  similar 


INFLUENCE  OF  TEMPERATURE  UPON  LIFE-PROCESSES     423 


characteristic  distinguished  the  temperature-coefficient  of  the  time 
consumed  in  the  pupal  stage  of  development: 


Temperature. 
13.45°     . 
15.55°     . 
17.0° 
18.8° 
20.9° 
23.65°     . 
27.25°     . 
32.7°       . 
32.95° 


Hours  spent  in  pupal 
condition. 


Temperature-coefficient 
per  10°  C. 


1116. 
742. 
593. 
439.6 
320. 
234.1 
172.5 
137.9 
134.25 


6.2 


4.9 


2.6 


1.5 


We  may  infer  that  the  time  spent  in  the  pupal  stage  depends  upon 
the  extent  of  tissue-oxidation  which  has  occurred. 

By  employing  curarized  frogs  and  decerebrated  turtles  Krogh  was 
also  enabled  to  investigate  the  effect  of  temperature  upon  the  Tissue - 
respiration  of  these  animals.  The  values  of  the  coefficients  obtained 
lay  between  2  and  4,  but  the  values  were  not  found  to  be  so  greatly 
affected  by  the  position  on  the  temperature-scale  of  the  temperature- 
range  employed  as  in  the  case  of  the  insect-larvae. 

The  influence  of  temperature  upon  the  Rate  of  Development  of 
organisms  is  again  of  a  similar  character.  Thus  Hertwig  has  investi- 
gated the  influence  of  temperature  upon  the  time  taken  to  reach  seven 
different  arbitrarily  chosen  stages  of  development  of  the  larvae  of  a 
frog,  Rana  fusca.  The  following  were  the  results  obtained: 


Temperature-coefficient  for  10°. 

Temperature- 

interval. 

Stage  I. 

Stage  II. 

Stage  III. 

Stage  IV. 

Stage  V. 

Stage  VI. 

Stage  VII. 

2.  5°  to     6° 

10. 

13. 

15. 

14. 

6.°    to  15° 

2.6 

2.6 

2.5 

2.6 

3.1 

3.5 

4.5 

10.°    to  20° 

2.9 

3.3 

3.2 

2.9 

3.5 

3.4 

3.3 

20.°    to  14° 

1.5 

1.4 

2.0 

2.0 

2.0 

2.0 

1.8 

We  have  seen  that  the  rate  of  development  in  the  pupal  stage  of 
insects  and  the  rapidity  of  their  basal  metabolism  are  very  similarly 
influenced  by  temperature,  so  that  we  may  infer  with  probability  that 
oxidations  determine  the  duration  of  this  period  of  development.  This 
is  not  the  case  in  the  earliest  stages  of  development,  however,  for 
Loeb  and  Wasteneys  have  investigated  the  influence  of  temperature 
upon  the  time  which  elapses  between  insemination  and  the  first  cell- 
division  in  sea-urchin  eggs  and  have  compared  with  this  the  effect  of 
the  same  temperatures  upon  the  oxygen-consumption  of  the  eggs. 
The  two  sets  of  temperature-coefficients  are  unmistakably  of  the 
magnitude  of  the  coefficients  of  chemical  reactions,  but  they  are  very 
diversely  affected  by  alteration  of  the  position  of  the  temperature- 
range,  as  the  following  figures  show: 


424    PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 


Temperature- 
interval. 


Temperature-coefficient  of 
rate  of  segmentation  in : 


3° 

to  13° 

4° 

to  14° 

5° 

to  15° 

7° 

to  17° 

8° 

to  18° 

9° 

to  19° 

10° 

to  20° 

11° 

to  21° 

12° 

to  22° 

13° 

to  23° 

15° 

to  25° 

16° 

to  26° 

17.5° 

to  27.  5 

20° 

to  30° 

Slronglycentrotu  s  . 
3.91 

Arbacia. 

......      3.88 
.      ...      .      3.52 
.      .      .     3.27          , 

7.3 

-'!      '.     2.04 
.      1.90 

6.0 
4.7 
3.8 

1.74 


3.3 
3.1 
2.8 
2.5 
2.6 
2.2 
1.7 


Temperature-coefficient 
of  rate  of  oxidation  in : 

Arbacia. 
2.18 

2.16 
2.00 


2.17 


2.45 
2.24 

2.00 
1.96 


Not  only  oxidations,  therefore,  but  some  other  chemical  factors 
even  more  susceptible  to  temperature  change  are  involved  in  deter- 
mining the  rapidity  of  the  cell-divisions  in  the  early  stages  of  develop- 
ment. 

Other  forms  of  growth,  for  example  Regeneration,  as  A.  R.  Moore 
has  shown,  are  also  affected  by  temperature  to  the  extent  characteristic 
of  chemical  reactions. 

In  all  of  the  life-processes  hitherto  mentioned  the  general  order  of 
magnitude  of  the  temperature-coefficients  has  been  the  same.  When, 
however,  we  come  to  study  the  temperature-coefficients  for  the  Dura- 
tion of  Life  we  meet  with  a  startling  disparity  of  quantitative  effects, 
for  whereas,  for  example,  it  takes  a  rise  of  nearly  ten  degrees  to  double 
the  rate  of  the  heart-beat,  or  the  rate  of  respiratory  movements  or  the 
rate  of  cell-division  or  regeneration  or  tissue-oxidations,  yet  a  tempera- 
ture-elevation of  merely  one  degree,  as  J.  Loeb  has  shown,  serves  to 
halve  the  duration  of  life  of  fertilized  or  unfertilized  eggs  of  the  sea- 
urchin,  and  lowering  of  the  temperature  by  ten  degrees  prolongs  the 
life  of  the  organism  210,  that  is  to  say  over  a  thousandfold.  The 
temperature-coefficient  of  the  processes  underlying  the  thermal  death 
of  the  cells  is  therefore,  no  less  than  1000.  A.  R.  Moore  has  investi- 
gated the  influence  of  various  temperatures  upon  the  duration  of  life 
of  a  hydroid,  Tubularia  crocea,  judging  viability  by  the  retention  of  the 
power  of  regeneration.  The  following  are  illustrative  results: 


Temperature. 

25° 
26° 
27° 
28° 
29° 
30° 
31° 
32° 
33° 
34° 
35° 
36° 


Dura 

55  to 
25  to 
15  to 
7  to 
130  to 
50  to 
30  to 
14  to 
7  to 
3  to 
2  to 
Uto 

•Aon  of  life. 
60  hrs. 

Coef 

IV 

.      .      .      2.0 

icient  for  tempe 
interval  of: 

30     "          *      * 

.      .     1.7 

18    "         '      ' 

.      .     2.2 

8     "         *      ' 

.      .     3.3 

140  mins.     ' 

.      .      2A\ 

60      "        '      ' 

40      «        '      '" 
15      "        '      '  - 
8      "        '      • 
4      "        '      ' 

.      .      1.6 
.      .      2.4 
.      .      1.9 
.      .      2.1 
1  4 

3      "        '      ' 
2      "        '      • 

.      .      1.5 

rature 
10°. 


3900 


485 


INFLUENCE  OF  TEMPERATURE  UPON  LIFE-PROCESSES     425 

It  is  obvious  that  here  we  are  dealing  with  a  phenomenon  of  quite 
a  distinct  nature  from  the  other  phenomena  of  life  which  we  have 
hitherto  being  considering,  and  the  question  immediately  suggests 
itself  whether  any  clue  exists  as  to  the  origin  of  this  remarkable 
susceptibility  to  temperature.  Now  on  comparing  the  temperature- 
coefficients  of  various  reactions  involving  Enzymes,  one  group  stands 
out  from  all  the  rest  by  reason  of  the  extraordinary  magnitude  of  the 
temperature-coefficients,  and  that  is  the  group  afforded  by  the  Auto- 
destruction  which  various  enzymes  undergo  in  solution.  The  following 
data  are  cited  after  Arrhenius : 

Numerical  value 
Nature  of  process.  of  /*. 

Hydrolysis  of  sugar  by  acids 25,600 

invertase 9,080 

Saponification  of  ethyl  acetate  by  NaOH 11,150 

triacetin  by  lipase 16,  700 


cottonseed  oil  by  lipase 

Digestion  of  gelatin  by  pepsin 

trypsin       .... 

"  egg-white  by  pepsin   .... 

Coagulation  of  milk  by  rennet        .... 

Spontaneous  destruction  of  trypsin  in  solution 

"  "  pepsin  in  solution 


7,540 
10,750 
10,570 
15,570 
20,650 
62,034 
75,600 


rennet  in  solution     .      ......      90,000 

vibriolysin  in  solution    ......     128,000 

tetanolysin  in  solution  ......     162,000 

hemolysin  in  solution     .      .      .      .  %  .      .198,500 


The  relationship  between  the  value  of  M  in  the  equation: 


and  the  temperature-coefficient  for  the  ten-degree  interval  between 
20°  and  30°  C.  is  shown  in  the  following  table: 

A  temperature-  Corresponding  to  the 

coefficient  of  value  of 

2  ................  13,200 

10  ................  44,000 

100  ................  88,000 

1,000  .........   .......  132,000 

10,000  .........   .......  176,000 

It  is  evident  therefore  that  the  temperature-coefficient  of  the 
duration  of  life  corresponds  not  at  all  with  that  of  enzymatic  hydrol- 
yses,  but  it  is,  on  the  other  hand,  of  precisely  the  order  of  magnitude 
encountered  in  the  autodestruction  of  enzymes  or  of  specific  anti- 
bodies. It  is  to  the  destruction  of  enzymes,  consequently,  that  we 
may  attribute  the  thermal  death  of  organisms  excepting  in  those  cases, 
as  in  spores  of  seeds,  in  which  the  essential  tissue-enzymes  are  thermo- 
stabile  and  the  temperatures  required  to  kill  the  tissue  are  those  which 


426    PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

suffice  to  coagulate  Proteins.  For  example,  Goodspeed,  who  investi- 
gated the  thermal  death  of  barley-seeds,  exposed  them  to  temperatures 
ranging  from  55°  to  70°  C.  and  obtained  a  temperature-coefficient  of 
10  for  the  duration  of  life,  a  coefficient  which  is  very  close  to  the  value 
8  obtained  by  Chick  and  Martin  in  estimating  influence  of  tempera- 
tures of  similar  magnitude  upon  the  rate  of  coagulation  of  Hemoglobin. 

The  remarkable  disparity  between  the  effects  of  temperature  upon 
the  Life-duration  and  the  Development  of  organisms  has  been  applied 
by  Loeb  to  the  explanation  of  what  would  otherwise  be  an  exceedingly 
puzzling  fact,  namely  the  extraordinary  density  of  the  population  of 
the  polar  seas.  In  his  account  of  the  Valdivia  expedition,  Chun1  calls 
especial  attention  to  the  quantitative  difference  in  the  surface  fauna 
and  flora  of  polar  and  temperate  or  tropical  regions :  "  In  the  icy  water 
of  the  Antarctic,  the  temperature  of  which  is  below  0°  C,  we  find  an 
astonishingly  rich  animal  and  plant-  life.  The  same  condition  with 
which  we  are  familiar  in  the  Arctic  seas  is  repeated  here,  namely,  that 
the  quantity  of  plankton  material  exceeds  that  of  the  temperate  and 
warm  seas."  And  again,  in  describing  the  pelagic  fauna  in  the  region 
of  the  Kerguelen  Islands  he  states:  "The  ocean  is  alive  with  trans- 
parent jellyfish,  Ctenophores  (Eolina  and  Callianira)  and  of  Siphono- 
phore  colonies  of  the  genus  Agalina." 

This  observation,  which  has  been  repeatedly  made  by  Arctic  and 
Antarctic  travellers,  would  appear  paradoxical  in  consideration  of  the 
effect  of  temperature  upon  development,  for  the  rate  of  development 
of  organisms  is,  as  we  have  seen,  halved  or  even  reduced  to  a  greater 
extent  by  a  drop  of  10°  C.  in  temperature.  When,  however,  we  reflect 
that  the  duration  of  life  of  these  slowly  developing  organisms  is  pro- 
longed a  thousandfold,  the  density  of  the  polar  population  becomes 
explicable,  for  the  net  result  of  these  opposed  effects  would  be  a  great 
increase  in  the  number  of  surviving  individuals  and  in  the  number  of 
successive  generations  simultaneously  inhabiting  the  cold  waters. 

The  temperature-coefficient  of  the  life-processes  which  we  have 
hitherto  considered  have  all  been  of  such  a  magnitude  as  to  clearly 
invite  the  supposition  that  the  velocities  of  the  phenomena  are  deter- 
mined by  the  rate  at  which  underlying  chemical  transformations  occur. 
We  now  come  to  a  life-phenomenon  of  peculiar  character  in  which  the 
testimony  of  the  temperature-coefficient  is  far  from  being  so  unequivo- 
cal, namely  the  Conduction  of  Stimuli  along  the  fibers  of  a  motor-nerve. 

The  influence  of  temperature  upon  the  rate  of  conduction  of  the 
nervous  impulse  was  first  investigated  by  S.  S.  Maxwell,  who  employed 
for  this  purpose  the  pedal  nerve  of  a  large  slug,  Ariolimax  columbianus. 
This  nerve  was  selected  on  account  of  its  considerable  length  and  the 
slowness  of  the  propagation  of  the  impulse  permitting  a  much  greater 
exactitude  of  measurement  than  is  possible  in  the  shorter  and  more 

1  Cited  after  J.  Loeb:  The  Mechanistic  Conception  of  Life. 


INFLUENCE  OF  TEMPERATURE  UPON  LIFE-PROCESSES     427 

rapidly  conducting  sciatic  nerves  of  a  frog.     The  following  table 
summarizes  the  results: 


Temperature-interval . 
-0.5°to     9.5° 
0.°    to  10.° 
1.°    to  11.° 
to  13.° 
to  15.° 
to  16.° 
to  19.° 
to  21.° 
11.5°to  21.5° 
12.5°to  22.5° 
13.°    to  23.° 
14.°    to  24.° 
16.°    to  26.° 


Temperature-coefficient. 
.  .  2.14 
.79 
.98 
.07 
.29 
.57 
.32 
.47 
.54 
.67 
.65 
.32 
.81 


It  will  be  seen  that  almost  the  only  coefficients  approaching  the 
numerical  value  of  2  are  those  obtained  at  the  lowest  temperature- 
ranges,1  nor  is  this  a  peculiarity  of  the  type  of  nerve-fiber  employed  by 
Maxwell,  for  the  later  investigations  of  Lucas  and  Gantor  on  the 
transmission  of  impulses  in  the  motor-nerves  of  frogs  bear  similar 
testimony.  The  following  are  the  results  obtained  by  Gantor: 


Temperature-coefficient  for  10°  interval. 
Experimental  series  number. 


Temperature. 

1 

2 

3 

4 

5 

0.        .      . 

. 

2.35 

.86 

2.5     .      . 

2.28 

.77 

5.        .      . 

.     2.03 

1.79 

.79 

1.97 

2.09 

7.5     .      . 

.      1.82 

1.64 

L.65 

1.95 

1.51 

10 

1  79 

1.60 

57 

1.95 

1  59 

12.5    '.    V 

.      1.66 

1.53 

]62 

1.81 

1.75 

15 

1  59 

1.48 

.55 

1.77 

1.67 

17  5     . 

.50 

1.68 

20. 

1.47 

1.61 

Average. 


1.87 
1.77 
1.71 
1.64 
1.61 


These  coefficients  are  intermediate  in  value  between  those  usually 
obtained  in  physical  phenomena  and  those  which  may  characterize 
chemical  transformations.  We  are  therefore  led  tovsuspect  that  physi- 
cal events  play  a  large  part  in  determining  the  rate  of  transmission  of 
nervous  impulses.  This  view  is  rendered  the  more  probable  by  the 
historical  difficulty  which  has  been  encountered  in  demonstrating  the 
existence  of  any  metabolic  changes  in  nerve-fibers  or  their  enhance- 
ment by  stimulation,  and  while  the  recent  results  of  Tashiro  demon- 
strate a  minute  evolution  of  carbon  dioxide  from  excised  nerves,  it 
cannot  be  regarded  as  proved  that  this  metabolic  activity  is  very  closely 
associated  with  the  conduction  of  the  stimulus.  It  may,  rather,  be 
concerned  with  the  maintenance  of  the  nutrition  or  repair  of  the  nerve, 
and  the  inability  of  nerve-fibers  to  display  fatigue  on  repeated  stimula- 
tion lends  strong  encouragement  to  this  view,  for  evidently  no  material 

1  A  small  number  of  coefficients  exceeding  2  are  attributed  by  Maxwell  to  experi- 
mental errors  and  are  not  included  in  the  above  averages. 


428    PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATTON 

is  used  up  in  consequence  of  their  excitation.  On  the  other  hand, 
Carlson's  experiments  with  the  heart  ganglion  of  Limulus  and  the 
above-cited  experiments  on  the  effects  of  heating  the  respiratory  center 
show  that  chemical  changes  play  a  predominant  role  in  the  activities 
of  Nerve  Cells,  and,  as  a  matter  of  fact,  the  consumption  of  oxygen 
by  the  brain  is  very  large,  and  it  is  the  first  tissue  to  suffer  from  lack  of 
oxygen,  indicating  a  very  high  level  of  metabolic  activity  in  the  cellular 
elements  of  the  nervous  system.  The  conducting  fibers  and  the  nerve- 
cells  from  which  they  issue  stand  therefore  in  sharp  contrast  to  one 
another  in  respect  to  the  metabolic  foundations  of  their  functional 
activity,  and  we  are  thus  led  to  recall  the  fundamental  difference 
between  their  susceptibilities  to  the  various  classes  of  chemical  stimu- 
lants to  which  reference  has  been  made  in  a  previous  chapter.  Nerve- 
fibers  are  powerfully  stimulated  by  salts  which  precipitate  calcium, 
nerve-cells  are  insensitive  to  these  reagents.  Nerve-cells  are  stimu- 
lated by  a  variety  of  specific  substances,  by  polyphenols  and  by 
Creatine,  for  example,  to  which  nerve-fibers  are  indifferent. 

The  phenomena  of  Muscular  Contraction  and  the  change  which  trans- 
forms the  nervous  impulse  into  a  muscular  stimulus  at  the  myoneural 
junctions  are,  it  would  appear,  conditioned  in  their  speed  by  underlying 
chemical  reactions.  Thus  Burnett  has  determined  the  influence  of 
temperature  upon  the  Latent  Period  of  indirect  muscular  stimulation 
(i.  e.,  through  the  intermediation  of  a  motor-nerve)  and  finds  that  the 
period  consumed  in  the  transformation  of  the  nervous  into  the  muscular 
stimulus  is  halved  or  even  more  reduced  by  a  rise  of  ten  degrees  in  the 
temperature.  Similarly  the  changes  involved  in  the  stimulation  of 
Sensory  Nerve  Endings  are  determined  by  chemical  factors,  since  T.  E. 
Moore  has  shown  that  the  temperature-coefficient  of  the  reaction  to 
cutaneous  stimulation  by  heat  is  of  the  chemical  magnitude.  Corre- 
sponding with  these  facts  we  find  that  nerve-endings  readily  undergo 
fatigue.  We  have  seen  that  the  rate  of  the  heart-beat  is  doubled  or 
more  than  doubled  by  a  rise  of  10°  and  the  same  thing  has  been  found 
to  be  true  for  other  rhythmic  muscular  contractions.  The  rate  of 
conduction  of  the  Action-current  in  Muscles,  however,  appears,  from 
the  investigations  of  Lucas,  to  be  a  process  analogous  to  the  conduc- 
tion of  a  nervous  impulse,  comparatively  little  affected  by  temperature 
(coefficient  from  1.45  to  1.65). 

It  is  a  general  characteristic  of  Photochemical  Reactions,  and  a  pecu- 
liarity which  distinguishes  them  from  all  other  types  of  chemical 
transformation,  that  they  are  practically  unaffected  by  temperature, 
the  temperature-coefficients  being  usually  unity,  and  at  any  rate  not 
in  excess  of  the  magnitudes  commonly  obtained  in  purely  physical 
phenomena.  This  being  the  case  it  is  a  very  significant  fact  that  the 
temperature-coefficients  of  the  phenomena  induced  by  light  in  living 
organisms  are  usually  high  and  distinctly  of  the  order  indicating  the 
involvement  of  chemical  reactions  of  the  ordinary  type.  Thus  the 
phototropic  bending  induced  by  light  in  Avena  sativa  has  been  shown 


INFLUENCE  OF  LIGHT  UPON  LIFE-PROCESSES  429 

by  de  Vries  to  be  increased  from  three  to  five  times  in  velocity  by  a 
rise  of  10°  in  temperature.  Still  more  remarkable  is  the  fact  that  the 
Assimilation  of  Carbon  Dioxide  by  green  plants  in  sunlight  which 
underlies  the  Photosynthesis  of  Carbohydrates  is  also  doubled  or  more 
than  doubled  by  a  rise  of  ten  degrees  in  temperature;  the  following 
results  are  compiled  from  the  measurements  of  Gabrielle  Matthsei,  the 
absorption  measured  being  that  of  leaves  of  Prunus  laurocerasus 
exposed  to  gaslight  of  constant  intensity: 

Carbon  dioxide         Temperature-coefficient 
Temperature.  assimilated.  per  10°  C. 

-6° 0.2  28.7 

0°  1.75  2.40 

10°  4.2  2.12 

20°  8.9  1.76 

30°  15.7  1.81 

37°  23.8  0.23 

40.5° 14.9 

Heat-injury  already  appears  at  30°,  but  below  this  temperature  the 
coefficients  clearly  indicate  that  the  rate  of  assimilation  is  not  deter- 
mined by  the  photochemical  process  but  by  a  reaction  of  the  ordinary 
type.  These  results  may  be  interpreted  by  supposing  that  the  photo- 
chemical reaction  (transformation  of  CO2  and  H2O  into  formaldehyde) 
is  retarded  by  its  product,  and  that  the  speed  of  photosynthesis  is 
therefore  determined  by  the  rate  at  which  this  product  is  removed  by 
a  secondary  reaction  (condensation  of  formaldehyde  into  glucose). 


THE  INFLUENCE  OF  LIGHT  UPON  LIFE-PROCESSES. 

Photosensitive  Substances  are  of  very  widespread  occurrence  in  living 
tissues.  This  is  evidenced  by  the  fact  that  the  effects  of  light  upon 
organisms  are  not  by  any  means  confined  to  the  specialized  cells  which 
comprise  the  visual  organs  in  the  higher  metazoa.  The  synthesis  of 
Carbohydrates  in  plants  is  brought  about  by  the  action  of  sunlight  upon 
vegetable  tissues  which  contain  Chlorophyll  or  some  analogous  pigment 
and  are  exposed  to  an  atmosphere  containing  carbon  dioxide,  but  quite 
independently  of  this,  light  additionally  exerts  an  effect  upon  the  proto- 
plasm of  the  cells  of  most  plants,  leading  to  a  bending  of  sessile  forms  or 
an  actual  migration  of  motile  forms  toward  or  away  from  the  source 
of  light,  a  phenomenon  known  as  Phototropism  or  Heliotropism.  This 
phenomenon  is  also  very  generally  displayed  by  animals,  and  the 
investigations  of  J.  Loeb  have  demonstrated  that  the  mechanism  of 
heliotropism  in  animals  and  in  plants  is  essentially  the  same,  nor  is  it 
invariably  associated,  even  in  animals,  with  the  possession  of  specific 
light-sensitive  organs.  For  many  of  the  unicellular  forms  of  life  sun- 
light is  very  definitely  toxic,  and  this  is  true  not  only  for  pigmented  but 
also  for  colorless  cells.  The  most  toxic  portion  of  the  spectrum  lies 
in  the  ultraviolet  region,  a  fact  which  bacteriologists  have  attempted 


430    PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

to  utilize  for  the  sterilization  of  water  and  milk;  a  difficulty  is  created, 
however,  by  the  deficient  power  of  ultraviolet  light  to  penetrate 
liquids,  which  necessitates  the  exposure  of  only  thin  layers  to  the  toxic 
rays. 

The  region  of  the  spectrum  which  is  most  efficient  in  causing  helio- 
tropic  movements  or  curvature  varies  in  different  species  of  animals 
and  plants.  Among  the  plants  the  rays  of  the  blue  end  of  the  spectrum 
are  usually  the  most  efficient.  Thus  Blaauw  has  determined  the  dura- 
tion of  exposure  to  light  derived  from  various  parts  of  the  carbon  arc 
which  is  necessary  to  induce  heliotropic  curvature  in  the  seedlings  of 
Avena.  The  following  are  his  results: 

Duration  of  illumination  Position  of  the  rays  in 

in  seconds.  the  spectrum. 

6300 •'..   ....   :   .   .   .   .   .  534  JUM 

1200 510  " 

120 499  « 

15 491  " 

5 487  " 

4 478  " 

3  .   .   .  .   .  .  .  .  .  .  ...  ...  ... 

4 ,   ...  466  " 

6 448  " 

The  maximum  effect  is  therefore  obtained  between  478  MM  and  466  ///z, 
that  is,  in  the  blue  region  of  the  spectrum.  Among  animal  forms 
Loeb  and  Wasteneys  have  found  that  Eudendrium  and  Arenicola  are 
similarly  affected  chiefly  by  the  blue  rays,  while  other  animals,  for 
example  the  crustaceans  and  insects  are  primarily  affected  by  the  rays 
lying  en  the  border  of  the  green  and  yellow.  Even  closely  allied  forms 
may,  however,  differ  very  decidedly  in  the  part  of  the  spectrum  which 
is  most  efficient  in  eliciting  heliotropic  curvature.  Thus  Loeb  and 
Wasteneys  have  observed  that  the  green  flagellate  Euglena  viridis 
is  most  affected  by  the  blue  rays  lying  between  X  =  470  and  480  ///*, 
while  the  closely  allied  flagellate  Chalmydomonas  pisiformis  is  especially 
sensitive  to  the  yellow-green  rays  in  the  neighborhood  of  X  =  534  w 

That  the  foundation  of  these  light  effects  resides  in  a  Photochemical 
Reaction  which  is  induced  within  the  organism  is  shown  by  the  applica- 
bility of  the  Bunsen-Roscoe  Law,  which  is  generally  characteristic  of 
photochemical  reactions  and  applies,  for  example,  to  the  blackening  of 
a  sensitized  photographic  plate  by  exposure  to  light.  This  may  be 
enunciated  as  follows:  The  chemical  effect  induced  by  light  is  pro- 
portional to  the  product  of  the  intensity  multiplied  by  the  duration 
of  illumination,  or  in  symbols: 

E  =  Kit 

where  E  is  the  extent  of  photochemical  transformation,  i  the  intensity 
of  the  light,  and  t  the  duration  of  the  illumination  and  K  a  propor- 
tionality-factor which  is  constant  for  the  particular  photochemical 
transformation  under  consideration. 


INFLUENCE  OF  LIGHT  UPON  LIFE-PROCESSES  431 

The  validity  of  the  Bunsen-Roscoe  law  in  the  heliotropism  of  organ- 
isms has  been  established  in  a  variety  of  investigations.  Thus  Blaauw 
has  determined  the  time  required  to  produce  heliotropic  curvature  in 
the  seedlings  of  Avena  sativa  by  varying  intensities  of  illumination 
with  the  following  results : 

Duration  of  Candle-meters 

Candle-meters.  illumination.  X  seconds. 

26.3 
20.6 
21.9 
18.6 
19.1 
16.2 
17.2 
18.3 
19.7 
22.4 
23.9 
21.6 
24.8 
27.5 
24.2 
21.8 
16.9 
18.9 
18.0 
24.7 
20.5 
22.8 
19.0 
19.8 
16.4 
26.5 

It  will  be  seen  that  the  product  of  the  intensity  into  the  duration  of 
the  illumination  approaches  the  constant  value  of  20,  and  indeed, 
when  one  considers  the  very  great  range  of  intensities  employed  and 
the  inherent  variability  of  living  material,  the  degree  of  constancy 
observed  is  really  astonishing.  A  remarkable  instance  of  the  applica- 
bility of  this  law  to  the  Heliotropism  of  animals  is  afforded  by  the 
experiments  of  Loeb  and  Wasteneys,  upon  the  polyps  of  Eudendrium. 
These  hydranths  are  exceedingly  variable  in  their  response  to  light  and 
it  was  accordingly  necessary  to  make  a  great  number  of  measurements 
and  treat  the  results  statistically.  The  polyps  were  exposed  to  three 
different  intensities  of  light,  a  light  of  definite  strength  being  stationed 
at  three  different  distances,  namely  25  cm.,  37.5  cm.  and  50  cm.  from 
the  organisms  and  they  were  exposed  to  the  light  for  such  periods  as 
to  render  the  product  i  X  t  a  constant.  Under  these  circumstances 
it  was  found  that  some  among  any  group  of  polyps  underwent  helio- 
tropic curvature,  while  others  did  not.  The  percentage  of  bent  polyps 
was  determined  in  each  case,  and  if  the  Bunsen-Roscoe  law  were  valid 
it  is  evident  that  these  percentages  should  be  the  same  for  all  three 
intensities  of  light,  i.  e.,  the  percentages  undergoing  bending  at  the 
distances  25,  37.5  and  50  cm.  from  the  light  should  be  the  same  or 
1  : 1  ;  1,  The  actual  ratios  were  determined  for  each  of  the  possible 


0.000439  

...       13      " 

0.000609       .      .      .      . 

...       10      " 

0.000855       .      . 

.      .      .         6      " 

0.001769.      .      . 

...         3      " 

0.002706.      .      .      .    ... 

.     100  minutes 

0  004773                        ' 

60        " 

0  01018   . 

30        " 

0  01640  .      . 

.      .       20        " 

0  0249     .      .      . 

.      .            15        " 

0.0498     

...        8 

0.0898     

...        4 

0.6156     .      .      .      .      : 

40  seconds 

1.0998     

...       25 

3.0281      

...        8 

5  456 

4        " 

8.453  

...         2 

18  94 

1        " 

45.05   

...          2/5            " 

308.7     

.         .         .           2/26           " 

511.4     

l/26           " 

1255.     •   .      .     

.'•••:  v«  " 

1902  

.      .      .       Vioo     " 

7905  

.         .         .          V«0       " 

13094.     ,  .      v-:    .      .      .      . 

.      .      .       Vsoo     " 

26520.        .:  „      .     -.      .      . 

.      .      .       Viooo    " 

432     PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

pairs  of  distances  and  they  differed  more  or  less  from  the  ideal  value 
of  unity,  but  the  average  of  a  large  number  of  these  ratios  differed  from 
unity  only  by  an  amount  commensurable  with  the  probable  error  of 
the  average.  The  following  are  their  results,  the  values  enclosed  in 
brackets  being  rejected  by  Chauvenet's  criterion  for  the  rejection  of 
extreme  variates.1 


Times  of  exposure  in  minutes. 

Ratio  of  percentage  of  hydranths  bending 
toward  light. 

25cm. 

37.5  cm. 

50  cm. 

25cm.;  37.5cm. 

25  cm.  ;  50  cm. 

37.5cm.;  50cm. 

15 

60 

1.50 

- 

20 

80 

.... 

1.30 

10 

22.5 

40 

1.20 

(3.08) 

(2.56) 

10 

22.5 

40 

0.94 

1.47 

1  .  55 

10 

22.5 

40 

1.57 

(2.30) 

(2.43) 

10 

22.5 

40 

1.43 

1.04 

0.94 

10 

22.5 

40 

0.76 

1.09 

1.47 

10 

22.5 

40 

1.05 

1.13 

0.90 

0.96 

10 

22.5 

40 

1.15 

.... 

0.99 

7 

15.75 

28 

0.84 

0.62 

0.74 

7 

15.75 

28 

1.70 

0.49 

0.58 

7 

15.75 

28 

0.85 

1.25 

1.35 

7 

15.75 

28 

(2.09) 

0.99 

1.08 

7 

15.75 

28 

1.14 

1.15 

0.55 

7 

15.75 

28 

0.44 

0.92 

0.44 

7 

15.75 

28 

1.52 

0.80 

0.61 

7 

15.74 

28 

0.59 

0.36 

0.70 

7 

15.75 

28 

0.48 

1.07 

0.31 

7 

15.75 

28 

1.00 

0.48 

1.80 

7 

15.75 

28 

0.69 

1.09 

0.81 

7 

15.75 

28 

1.26 

0.85 

1.09 

7 

15.75 

28 

0.86 

1.38 

0.85 

7 

15.75 

28 

0.70 

1.07 

1.59 

7 

15.75 

28 

0.77 

1.25 

7 

15.75 

28 

0.60 

\ 

Average*  . 

1   01 

fl  Q7 

0   Qfl 

Probable  err 

or*    .      .    .  ,. 

j.  .  \j  i 

U  .   »7  / 

U  .  t7O 

±0.05 

±0.04 

±0.06 

*  The  averages  and  probable  errors  given  are  those  recalculated  by  the  authors 
since  the  original  article  was  published. 

It  is  a  general  law  of  Photochemical  Action  that  only  those  rays  are 
effective  which  are  absorbed  by  the  system  in  which  the  reaction 
occurs.  Visible  light-rays  are  not,  as  a  general  rule,  selectively  ab- 
sorbed by  protoplasm  and  hence  their  action  is  usually  confined  to  or 
exerted  reflexly  through  specialized  pigmented  areas  which  constitute 
the  receptive  elements  of  optical  sense-organs.  White  light  which  is 
not  toxic  for  the  majority  of  tissues  may  be  rendered  toxic,  as  L.  Loeb 
has  shown,  by  impregnating  the  tissue  with  certain  dyes,  particularly 
Eosin,  which  in  such  cases  acts  as  the  photochemical  absorbent  or 
sensitizer.  Ultraviolet  Light,  however,  is  universally  toxic  even  for 

1  W.  Chauvenet:  A  Manual  of  Spherical  and  Practical  Astronomy,  Philadelphia, 
1891,  vol.  2,  p.  558. 


INFLUENCE  OF  LIGHT  UPON  LIFE-PROCESSES  433 

colorless  organisms,  and  since  this  toxicity  presumably  depends 
upon  and  is  attributable  to  photochemical  reactions,  the  question 
presents  itself:  To  which  constituent  of  the  protoplasm  are  we  to 
attribute  the  selective  absorption  of  these  rays  which  we  may  presume 
to  be  the  necessary  precedent  to  their  photochemical  activity? 

It  was  pointed  out  over  forty  years  ago  by  Soret  that  the  majority 
of  proteins  exhibit  a  well-marked  absorption-band  in  the  ultraviolet 
part  of  the  spectrum.  In  seeking  for  the  origin  of  this  absorption- 
band  Soret  found  that  it  is  especially  well  exhibited  by  solutions  of 
Tyrosine  and  therefore  referred  it  to  the  tyrosine  radical  in  the  protein 
molecule.  These  observations  have  been  extended  by  Kober,  who  has 
carried  out  a  spectrographic  examination  of  solutions  of  the  various 
Amino-acids  which  are  the  end-results  of  protein  hydrolysis  and  of 
certain  Polypeptides.  Kober  has  confirmed  the  existence  of  an  absorp- 
tion-band in  the  ultra-violet  in  solutions  of  tyrosine,  and  finds  that  a 
similar  band  is  exhibited  by  solutions  of  Phenylalanine.  The  other 
amino-acid  constitutents  of  the  protein  molecule  exhibit  only  general, 
i.  e.,  non-selective  absorption  in  the  ultraviolet  spectrum. 

The  possibility  is  thus  indicated  that  the  tyrosine  and  phenylalanine 
radicals  of  the  proteins  constitute  the  optical  sensitizers  which  render 
living  cells  susceptible  to  the  toxic  action  of  ultraviolet  light.  If  this 
were  the  case,  then  passage  of  the  light  through  solutions  of  proteins  or 
the  aromatic  amino-acids  should,  by  absorption  of  the  toxic  ray,  to  a 
greater  or  less  extent,  deprive  the  light  of  its  toxicity  for  protoplasm. 
This  possibility  has  been  investigated  by  Harris  and  Hoyt,  who  have 
found  that  the  passage  of  ultraviolet  light  through  protein  or  peptone 
solutions  partially  detoxicates  it,  while  passage  through  solutions  of 
Cystine,  Tyrosine  or  Amino -benzole  Acid  has  a  remarkable  effect  in 
shielding  the  organisms  from  injury.  Other  dissolved  substances  such 
as  sugar,  urea,  alanine,  glycocoll,  etc.,  were  found  to  be  devoid  of  pro- 
tective power.  Leucine  undergoes  decomposition  when  exposed  to 
ultraviolet  light  and  it  exerts  a  certain  measure  of  protection.  The 
following  are  illustrative  results,  the  light  from  a  Cooper-Hewitt  ultra- 
violet light  being  passed  through  the  solution  contained  in  a  quartz 
beaker  before  reaching  the  organisms  (Paramoecia)  suspended  in  dis- 
tilled water  below  the  beaker : 

Average  determination- 
Solution,  period,  seconds. 

Water 130 

1 .  0  per  cent,  al  anine 130 

1 . 0  per  cent,  glycocoll .  130 

1 .  0  per  cent,  aspartic  aci  d 130 

1 . 0  per  cent,  glutamic  acid 135 

1 . 0  per  cent,  leucine .  250 

0 . 5  per  cent,  tyrosine 420 

1 .  0  per  cent,  amino-benz-oic  acid 2400 

0 . 5  per  cent.  NaOH 150 

1 . 0  per  cent.  NaOH 170 

1 .  0  per  cent,  glutamic  acid  in  1  per  cent.   NaOH 200 

1.0  per  cent,  cystine  in  0.5  per  cent.  NaOH 1200 

1 . 0  per  cent,  tyrosine  in  0.  2  per  cent.  NaOH     .      .      .          Unaffected  after  40 

minutes'  exposure, 
28 


434    PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

The  absorption  of  ultraviolet  rays  by  tyrosine  has  been  found  by 
Kober  to  be  markedly  increased  by  an  alkaline  reaction  and,  as  the 
above  results  show,  the  detoxication  of  the  ultraviolet  light  by  tyrosine 
solutions  is  also  very  greatly  increased  by  an  alkaline  reaction. 

The  results  of  Harris  and  Hoyt  are  thus  in  harmony  with  the  view 
that  the  susceptibility  of  protoplasm  to  ultraviolet  light  is  conditioned 
by  the  selective  absorption  and  consequent  "activation  of  the  toxic 
rays  by  the  aromatic  ami  no-acid  radicals  of  the  proteins.  These 
results  have  a  practical  as  well  as  a  theoretical  bearing,  for  they  imply 
that  fluids  containing  proteins  would  be  much  more  difficult  to  sterilize 
with  ultraviolet  light  than  water,  owing  to  the  protective  action  of 
the  proteins  in  the  fluid  through  which  the  light  has  to  pass  before 
it  impinges  upon  the  protoplasm  of  the  infecting  organisms. 

THE  STORAGE  OF  POTENTIAL  ENERGY:  THE  PHOTO- 
SYNTHESIS OF  CARBOHYDRATES. 

The  leaves  and  other  chlorophyll-containing  organs  of  green  plants 
absorb  Carbon  Dioxide  from  the  atmosphere  and  simultaneously  liberate 
an  equal  volume  of  oxygen.  The  carbon  which  is  thus  retained  is 
built  up  into  the  tissues  or  reserve-materials  of  the  plant,  appearing 
chiefly  in  the  form  of  Carbohydrates  which  accumulate  very  rapidly 
during  active  assimilation. 

The  process  of  carbon  dioxide  assimilation  by  green  plants  takes 
place  only  in  the  light  and  in  the  presence  of  Chlorophyll  or  related 
pigments.  Within  certain  limits  the  rate  of  absorption  is  proportionate 
to  the  intensity  of  the  illumination  of  the  leaf,  and  to  the  percentage 
of  carbon  dioxide  in  the  atmosphere.  Not  all  parts  of  the  spectrum 
are  equally  efficient  in  promoting  this  process,  the  red  rays  between  B 
and  C  causing  the  most  rapid -assimilation  while  the  activity  of  the  rays 
between  D  and  E  on  the  Frauenhofer  scale  is  a  minimum,  and  there  is  a 
second  maximum  in  the  violet,  beyond  R  This  was  first  shown  in  a 
most  ingenious  manner  by  Engelmann.  The  Aerobic  Bacteria  require 
the  presence  of  free  oxygen  to  display  motility.  If  some  green  algae 
and  aerobic  bacteria  be  imprisoned  together  under  an  air-tight  cover- 
glass  and  kept  in  the  dark,  the  free  oxygen  is  soon  consumed  and  the 
bacteria  become  motionless.  If  the  cell  is  now  exposed  to  light  the 
algae  decompose  carbon  dioxide,  setting  free  oxygen,  and  the  bacteria 
become  motile  again.  Exposure  of  the  cell  in  different  parts  of  the 
spectrum  yielded  the  above-quoted  results. 

The  assimilation  of  carbon  dioxide  by  green  plants  is  the  foundation 
of  the  existence,  not  only  of  the  plants  themselves,  but  of  the  animal 
world.  The  radiant  energy  of  the  sun  which  is  in  this  manner  stored 
up  in  the  tissues  of  the  green  plants,  reappears  at  the  other  extremity 
of  the  life-process  as  the  heat  or  muscular  energy  and  mechanical  work 
of  an  animal.  A  very  striking  peculiarity  of  living  material  also 
originates  in  this  process,  for  while  the  components  of  protoplasm  are, 


STORAGE  OF  POTENTIAL  ENERGY  435 

as  a  rule  optically  active,  i.  e.,  rotate  the  plane  of  polarized  light  to  the* 
right  or  left,  the  products  of  laboratory-syntheses  and  those  substances 
in  nature  which  have  never  passed  through  the  life-cycle  (and  some  of 
those  which  have  done  so)  are  optically  inactive.  It  is  true  that  we 
can  decompose  a  racemic  and  optically  inactive  mixture  into  optically 
active  parts  by  utilizing  the  selective  enzymatic  activities  of  Yeasts, 
or,  as  Pasteur  did,  we  may  sort  out  large  crystals  by  hand  into  two 
kinds  possessing  equal  and  opposite  rotatory  powers,  but  it  will  be 
observed  that  all  of  these  processes  involve  the  intrusion  of  a  living 
agent.  According  to  the  view  of  Byk,  optical  activity  originated  in 
the  earth  through  the  circular  polarization  of  light  which  occurs  when 
light  is  reflected  from  the  surface  of  the  sea.  If,  on  the  other  hand,  we 
revert  to  Arrhenius'  theory  of  the  origin  of  life  upon  the  earth,  we  may 
suppose  that  optical  activity  was  transmitted  to  this  planet  by  Bacterial 
Spores  floating  in  interstellar  space.  However  this  may  be,  the 
phenomenon  of  optical  activity  is  at  present  a  distinguishing  charac- 
teristic of  the  components  of  living  matter,  and  it  originates  in  the  very 
first  step  in  the  life-cycle,  for  the  carbohydrates  which  result  from  the 
photosynthetic  activities  of  plants  are  optically  asymmetrical. 

Notwithstanding  the  fact  that  the  immediate  connection  between 
the  assimilation  of  carbon  dioxide  by  green  plants  and  the  appearance 
of  carbohydrates  has  long  been  understood,  the  intermediate  products 
which  are  formed  in  the  process;  the  various  stages  which  link  the 
absorption  of  carbon  dioxide  to  the  appearance  of  starch  or  sugars  in 
the  tissues,  have  long  been  sought  for  in  vain.  The  classical  theory, 
proposed  by  Baeyer  in  1 870,  is  that  the  carbon  dioxide  is  first  reduced 
to  Formaldehyde 

CO2     +     H2O     ->    HCHO     +     O2 

and  that  the  formaldehyde  which  is  thus  formed  is  subsequently  con- 
densed to  a  hexose: 

6HCHO      =     C6Hi2O6 

If  this  view  is  correct  then  we  should  expect  to  find  formaldehyde 
among  the  constituents  of  the  green  plants  when  engaged  in  active 
assimilation.  Very  many  attempts  have  been  made  to  establish  the 
presence  of  formaldehyde  in  the  tissues  of  plants  and  they  cannot  yet 
be  said  to  have  yielded  any  very  definite  information.  Several  excep- 
tional difficulties  attach  to  this  investigation.  In  the  first  place  it  is 
certain  that  if  formaldehyde  occurs  in  green  leaves  at  all  it  is  never 
present  except  in  very  minute  amounts.  Indeed  it  is  essential  that  this 
should  be  so,  because  formaldehyde  is  a  very  powerful  protoplasmic 
poison  and  the  accumulation  of  any  amount  in  excess  of  a  minute  trace 
would  result  in  the  complete  arrest  of  protoplasmic  activities.  Thus 
Elodea  canadensis  is  cited  as  a  form  which  is  exceedingly  resistant  to 
the  toxic  action  of  formaldehyde,  yet  it  will  only  withstand  a  0.001 
per  cent,  solution. 


436     PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

We  must  expect  to  find  formaldehyde  in  vegetable  tissues,  if  it 
occurs  therein  at  all,  therefore,  only  in  minute  traces.  Now  although 
we  possess  very  sensitive  reagents  for  aldehydes,  yet  these  do  not  as  a 
general  rule  exclude  the  possibility  of  much  more  complex  aldehydes 
than  formaldehyde  being  present  and  yielding  the  reaction.  Even 
Proteins  and  many  other  tissue-constituents  will  yield  reactions  indica- 
tive of  an  aldehyde-grouping.  This  would  not  perhaps  constitute  an 
insurmountable  difficulty  if  it  were  not  for  the  fact,  as  we  have  seen, 
that  the  aldehyde  we  are  seeking  to  identify  is  at  the  most  only  present 
in  minute  traces. 

Another  way  of  attacking  the  problem  might  appear  to  be  feasible, 
namely  that  of  extracting  Chlorophyll  from  green  plants  and  utilizing 
its  light-activating  properties  to  bring  about  the  synthesis  of  for- 
maldehyde in  laboratory-glassware,  apart  from  the  complications  and 
secondary  reactions  which  attend  the  process  in  living  tissues.  Numer- 
ous attempts  to  accomplish  this  have  failed.  According  to  Usher  and 
Priestley,  however,  the  source  of  failure  has  resided  in  the  employment 
of  comparatively  thick  layers  of  the  chlorophyll  solution.  If  we  blow 
carbon  dioxide  through  a  test-tube  or  flask  filled  with  chlorophyll  and 
exposed  to  light,  we  cannot  expect  to  observe  much  photosynthesis, 
because  the  most  superficial  layers  of  the  chlorophyll  solution  will 
absorb  all  of  the  active  light-rays  and  transmit  to  the  underlying 
solution  only  those  which  are  chemically  inactive.  In  the  living  plant 
the  chlorophyll  is  disposed  quite  differently.  Here  we  observe  that 
pigment  is  confined  to  exceedingly  thin  layers  at  the  surfaces  of  a  series 
of  bodies  known  as  the  Chloroplasts  in  which  active  photosynthesis 
can  be  shown  to  be  proceeding  during  illumination.  Usher  and 
Priestley  have  sought  to  imitate  this  architecture  of  the  photosynthetic 
apparatus,  by  painting  the  surfaces  of  plates  of  gelatin  with  a  thin 
layer  of  chlorophyll  and  then  blowing  carbon  dioxide  over  them  and 
exposing  them  to  light.  Under  these  conditions  they  state  that  a 
comparatively  rapid  disengagement  of  oxygen  occurs,  the  surface  film 
becoming  wrinkled  and  distorted  by  the  accumulation  of  bubbles  of 
oxygen  below  it,  while  very  evident  quantities  of  formaldehyde  are 
found  in  the  underlying  gelatin.  The  accumulation  of  formaldehyde 
in  this  case,  as  contrasted  with  its  evanescence  in  the  tissues  of  plants 
they  refer  to  the  absence  of  the  enzymes  necessary  to  accomplish  the 
removal  of  the  formaldehyde  by  condensation,  which,  in  the  plants,  are 
present  in  the  underlying  substance  of  the  chloroplasts.  Against  this 
experiment  it  has  been  urged  by  several  investigators  that  the  presence 
of  formaldehyde  in  gelatin  jellies  is  very  difficult  to  establish,  since 
most  samples  of  gelatin  themselves  yield  a  very  pronounced  aldehyde- 
reaction.  Usher  and  Priestley,  however,  state  that  the  gelatin  which 
they  employed  was  free  from  aldehydes.  On  the  other  hand  the  syn- 
thesis of  formaldehyde  and  other  products  from  carbon  dioxide  and 
water  has  frequently  been  accomplished  without  the  intermediation  of 
chlorophyll  by  the  use  of  the  silent  electric  discharge,  and  by  exposure 


STORAGE  OF  POTENTIAL  ENERGY  437 

to  ultraviolet  light  or  to  sunlight  in  solutions  containing  salts  of 
Uranium. 

We  have  seen  that  the  rate  of  assimilation  of  Carbon  Dioxide  is 
governed,  not  primarily  by  the  velocity  of  the  photochemical  reaction, 
birt  by  the  velocity  of  a  subsequent  reaction  which  removes  its  products. 
This  is  shown  by  the  fact  that  the  temperature-coefficient  of  carbon- 
dioxide  assimilation  is  of  the  usual  chemical  magnitude  and  not  unity, 
as  would  be  the  case  in  a  purely  photochemical  process.  If  the  prod- 
uct of  the  photochemical  reaction  is  in  truth  formaldehyde,  as  Baeyer's 
hypothesis  assumes,  then  its  accumulation  would  very  evidently  be 
injurious  and  we  can  readily  understand  how  its  removal,  which 
presumably  does  not  require  the  agency  of  illumination,  would  be  an 
essential  condition  of  the  continuance  of  the  reaction  and  would  "  set 
the  pace"  of  the  whole  process.  It  is  not  certain,  however,  at  exactly 
what  stage  of  carbohydrate-synthesis  the  necessity  for  light  ceases. 
Thus  W.  Loeb  has  obtained  not  only  the  formation  but  also  the  partial 
polymerization  of  formaldehyde  with  the  silent  electric  discharge. 
The  fact  that  starch-formation  will  go  on  in  tubers  and  other  plant- 
tissues  which  are  not  exposed  to  the  light  throws  no  light  upon  this 
question,  for  the  starch  in  these  instances  is  not  formed  from  formalde- 
hyde but  from  hexoses  or  other  comparatively  complex  carbon  com- 
pounds. 

In  regard  to  the  nature  of  the  earliest  carbohydrate  to  arise  in 
photosynthesis  the  most  natural  supposition  would  appear  to  be  the 
formation  of  Glucose 

6HCHO      =     CeHizOe 

or  some  other  hexose,  since  this  synthesis  has  actually  been  performed 
in  the  laboratory.  As  a  matter  of  fact,  however,  there  is  much  evi- 
dence tending  to  show  that  the  first  carbohydrate  to  be  produced  in 
photosynthesis  is  actually  Cane-sugar  (sucrose).  This  view,  which 
was  first  put  forward  by  Brown  and  Morris,  has  received  very  strong 
support  from  the  investigations  of  Parkin.  This  observer  employed 
for  his  experiments  the  leaves  of  the  snowdrop,  Galanthus  nivalis, 
which  are  peculiar  in  that  they  do  not  form  Starch  during  photosyn- 
thesis, so  that  the  analyses  of  sugar-content  are  not  complicated  by 
the  possible  presence  of  sugars,  Maltose  or  Glucose,  derived  from  the 
hydrolysis  of  starch.  As  a  matter  of  fact  it  was  found  by  Parkin 
that  the  leaves  of  the  snowdrop  contain  only  three  carbohydrates, 
namely  Sucrose,  Fructose  (levulose)  and  Glucose.  Of  these  the  per- 
centages of  hexose  remain  very  constant  throughout  any  given  twenty- 
four  hours,  not  increasing  during  the  illumination  of  the  day,  nor 
decreasing  during  the  night,  while  the  percentage  of  sucrose  rapidly 
increases  during  the  day  and  decreases  decidedly  at  night.  Moreover 
the  proportion  of  sucrose  to  the  other  sugars  is  greatest  at  the  apical 
portions  of  leaves  where  assimilation  is  most  active,  and  decreases 
toward  the  base.  Two  interpretations  of  this  result,  however,  may 


438    PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

be  advanced.  The  cane-sugar  may  be  in  truth  the  first  sugar  to  be 
synthesized,  or,  on  the  other  hand,  glucose  may  be  the  first  sugar 
formed,  levulose  arising  from  it  by  a  transformation  which  can  be 
accomplished  in  laboratory-glassware,  and  the  constancy  of  the  hexose 
percentage  may  merely  mean  that  hexose  in  excess  of  this  amount  is 
condensed  to  cane-sugar  as  rapidly  as  it  is  formed.  It  should  be 
mentioned,  however,  that  the  formation  of  levulose  from  glucose  by 
alkalies  in  laboratory-glassware  is  accompanied  by  the  simultaneous 
formation  of  Mannose,  which  sugar  is  absent  from  foliage- leaves.  On 
the  other  hand  no  laboratory-method  of  directly  deriving  cane-sugar 
from  formaldehyde  has  yet  been  discovered. 

THE  CONVERSION  OF  CHEMICAL  INTO  MECHANICAL  ENERGY: 
THE  CHEMICAL  MECHANICS  OF  MUSCULAR  CONTRACTION. 

We  have  seen  that  upon  a  normal  mixed  diet  the  necessary  energy 
for  the  performance  of  muscular  work  is  derived  from  the  oxidation  of 
Carbohydrates  and  that  the  final  products  of  this  oxidation  are  carbon 
dioxide  and  water,  an  intermediate  stage  of  the  combustion  being  the 
formation  of  Lactic  Acid.  So  much  we  can  ascertain  by  methods  of 
direct  analysis.  If  we  desire,  however,  to  complete  the  story  of  the 
energy-cycle  which  begins  with  photosynthesis  in  the  plant,  and  cul- 
minates in  the  release  of  heat  and  mechanical  work  by  the  animal, 
purely  analytical  methods  will  not  suffice  and  we  are  impelled  to  seek 
additional  information  by  the  method  of  inference  from  indirect 
observation. 

Our  object  is  to  ascertain  the  nature  of  the  chemical  machine  which 
transforms  the  potential  energy  of  carbohydrates  into  muscular  work 
and  heat.  This  problem  divides  itself  into  two  parts,  namely  the 
question  of  the  nature  of  the  process  of  combustion  and  the  question 
of  the  means  of  transforming  the  energy  which  combustion  releases 
into  mechanical  work. 

In  respect  to  the  first  of  these  questions,  it  has  long  been  a  familiar 
fact  that  when  a  muscle  is  repeatedly  stimulated,  either  directly  or 
indirectly  through  its  motor-nerve,  the  first  few  contractions  gradually 
and  with  considerable  regularity  increase  in  height  until  they  reach  a 
maximum  for  a  given  strength  of  stimulus.  This  phenomenon  to 
which  the  name  of  "treppe"  or  the  "Staircase  Phenomenon"  was  given 
by  Bowditch,  has  been  the  subject  of  considerable  investigation  and 
conjecture.  Of  a  similar  nature  is  the  phenomenon  of  "Summation  of 
Stimuli,"  whereby  a  stimulus  of  strength  insufficient  to  give  rise  to  a 
response  when  it  is  first  applied,  may  be  made,  by  repetition,  to  elicit 
a  response. 

It  is  to  Waller  that  we  owe  the  suggestion  that  the  "staircase"  is, 
in  reality,  due  to  the  increased  production  of  carbon  dioxide  by  the 
contractile  or  conducting  tissue.  He  observed  that  small  amounts  of 
carbon  dioxide  augment  the  electrical  response  of  nerve-fibers  to 


CONVERSION  OF   CHEMICAL  INTO   MECHANICAL   ENERGY     439 

stimulation  and  that  a  short  tetanization  of  the  nerve  produced  a  pre- 
cisely similar  augmentation.  Lee  has  extended  this  idea  to  muscular 
tissues  and  he  has  pointed  out  that  the  action  of  the  products  of 
muscular  activity  upon  the  performance  of  muscular  work  is  two-fold 
producing  in  moderate  quantities  or  for  a  short  time  a  marked  increase 
in  the  irritability  and  working-power  of  the  muscle,  while  in  larger 
quantities  or  after  a  longer  period  of  action  they  produce  a  marked 
depression  or  "Fatigue*'  of  the  muscle,  ending  by  totally  preventing  the 
further  release  of  muscular  energy.  The  nature  of  the  products  which 
bring  about  these  results  has  been  established  by  Lee,  who  has  found 
that  perfusion  of  a  muscle  with  a  dilute  solution  of  Lactic  Acid  or  an 
acid  phosphate  increases  its  irritability  and  power  to  do  work,  while 
more  concentrated  solutions  of  the  same  substances  diminish  and 
finally  abolish  its  irritability  and  contractility.  Both  of  these  sub- 
stances are  known,  by  direct  estimation,  to  accumulate  in  a  muscle 
which  is  doing  work. 

If  we  consider  a  muscle  which  is  being  tetanized  by  rapidly  repeated 
stimuli,  it  is  evident  that  the  rate  at  which  the  muscle  is  doing  work 
may  be  regarded  as  an  expression  of  the  rate  at  which  the  underlying 
chemical  changes  are  taking  place.  During  the  initial  or  rising  part  of 
the  curve  of  tetanus  which  is  nearly  always  to  be  observed,  the  velocity 
of  the  underlying  chemical  changes  must  be  increasing.  During  the 
period  of  maximal  contraction  while  the  recording-lever  remains  at  a 
constant  level  it  is  evident  that  the  rate  of  doing  work  and  therefore 
the  velocity  of  the  underlying  chemical  change  are  practically  constant, 
during  the  third,  or  descending  part  of  the  curve  the  velocity  of  the 
chemical  changes  is  evidently  decreasing.  Similar  considerations 
apply,  of  course,  when  the  muscle,  instead  of  being  stimulated  at 
extremely  small  intervals,  is  being  stimulated  at  longer  intervals. 

The  chemical  changes  which  underlie  and  determine  muscular  con- 
traction are  of  such  a  character,  therefore,  that  one  or  more  of  the 
products  which  result,  first  accelerate  and  later  retard  the  process.  We 
are  familiar  with  many  chemical  reactions  of  this  type;  they  are  reactions 
in  which  one  of  the  products  acts  as  a  catalyzer  to  the  process  and  are 
therefore  designated  Autocatalyzed  Reactions.  Thus  in  the  hydrolysis 
of  Cane-sugar  by  neutral  boiling  water  small  quantities  of  mucic  acid 
are  developed  which  greatly  accelerate  the  inversion.  The  hydrolysis 
of  Methyl  Acetate  by  water  results  in  the  liberation  of  acetic  acid  which 
very  greatly  accelerates  the  hydrolysis.  The  hydrolysis  of  the  Ricino- 
leic  Acid  in  pulverized  castor-oil  beans  proceeds  at  first  very  slowly, 
and  then  with  great  rapidity,  the  acid  which  is  first  liberated  enhancing 
the  activity  of  the  lipase  in  the  macerated  tissues.  Instances  of  auto- 
catalytic  oxidations  are  afforded  by  the  spontaneous  oxidation  of  many 
Metals  and  organic  compounds  in  the  presence  of  oxygen  at  atmos- 
pheric temperature  and  pressure.  It  has  long  been  observed  that  in 
the  spontaneous  oxidation  of  these  substances  they  acquire  the  power 
of  inducing  oxidations  in  other  substances  which  are  not  spontaneously 


440     PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

oxidizable,  and  it  has  been  shown  that  this  action  is  due  to  the  forma- 
tion of  peroxides  which  catalyze  oxidations,  including  the  oxidation  of 
the  spontaneously  oxidizable  material  itself.  In  the  case  of  metals  the 
process  is  ultimately  brought  to  a  close  by  the  thickness  of  the  covering 
of  oxide  which  excludes  the  air.  To  preserve  metals  from  spontaneous 
oxidation,  therefore,  one  of  two  methods  should  be  adopted:  Either 
they  should  constantly  be  kept  clean  and  polished  to  avoid  the  accumu- 
lation of  catalyzers,  or  they  should  be  allowed  to  become  so  com- 
pletely tarnished  that  air  can  no  longer  penetrate  to  the  underlying 
metal.  The  intermediate  policy  of  sporadic  infrequent  polishing  leads 
to  maximal  loss  of  the  metal  by  oxidation. 

Very  many  instances  of  autocatalysis  are  afforded  by  the  spontaneous 
oxidation  of  fats  and  oils,  and  particularly  by  the  oxidation  of  the 
"Drying  Oils"  which  are  employed  in  paints  and  varnishes. 

It  is  a  general  characteristic  of  the  processes  of  Autocatalysis  that  they 
begin  relatively  slowly,  progressively  increase  in  velocity  to  a  maximum, 
and  then  fall  off  in  velocity  again  until  the  reaction  finally  ceases. 
The  cessation  of  the  reaction  may  be  due  to  the  exhaustion  of  the 
Substrate  or  material  undergoing  transformation,  as  for  example  in  the 
hydrolysis  of  cane-sugar,  or  it  may  be  due  to  the  back-pressure  of  the 
accumulated  products,  as  in  the  case  of  the  hydrolysis  of  methyl 
acetate.  In  general  the  autocatalyzers,  like  other  catalysts,  accelerate 
the  attainment  of  equilibrium  from  either  direction. 

The  underlying  combustion  which  releases  the  heat  and  mechanical 
energy  of  muscular  contraction  is  therefore  an  example  of  a  large  class 
of  chemical  transformations  which  produce  their  own  catalyzers.  Of 
the  various  stages  of  the  process  only  a  few  are  known,  but  among  the 
known  products  lactic  acid  and  carbon  dioxide  are  capable  of  identifica- 
tion as  direct  or  indirect  catalyzers  of  the  combustion. 

Our  knowledge  of  the  second  phase  of  the  problem  which  is  presented 
by  the  genesis  of  muscular  work  and  heat  is  still  more  fragmentary  and 
much  more  conjectural.  No  machines  of  the  ordinary  type  with  the 
details  of  which  we  are  familiar,  such  as  those  which  operate  by  gaseous 
or  liquid  pressures  and  mechanical  thrusts,  will  even  approximate  in 
characteristics  and  behavior  to  the  motile  mechanisms  of  living  proto- 
plasm. The  low  and  only  very  slightly  fluctuating  heat  of  combustion 
precludes  any  explanation  attributable  to  alternate  expansions  and 
contractions  due  to  heating  and  cooling.  Engelmann,  indeed,  has 
proposed  such  an  explanation!,  based  upon  the  supposition  that  intense 
heating  of  minute  particles  in  the  muscle-substance  may  occur  in  a 
number  of  circumscribed  foci.  He  has  pointed  out  that  a  number  of 
Doubly  Refracting  Substances,  such  as  catgut  or  India-rubber,  have  the 
unusual  property  of  contracting  when  they  are  heated,  and  he  assumes 
that  the  heat-energy  of  combustion  in  muscular  tissue  is  directly  trans- 
formed into  mechanical  work  by  transient  intense  heating  of  localized 
doubly  refracting  elements.  Many  objections  have  been  urged  against 
this  hypothesis  and  they  appear  in  our  present  state  of  knowledge  to 


CONVERSION   OF   CHEMICAL   INTO   MECHANICAL   ENERGY     441 

be  insurmountable.  The  objection,  for  instance,  that  living  matter  is 
destroyed  at  the  height  of  temperature  required  was  met  by  Engelmann 
by  supposing  that  the  elements  so  heated  only  form  a  very  small  pro- 
portion of  the  whole  contractile  tissue.  If  this  be  so,  then  they  cannot 
be  the  doubly  refractile  elements  which  we  perceive  under  the  micro- 
scope, for  these  form  a  very  large  proportion  of  the  whole.  The  foun- 
dation of  Engelmann's  analogy  between  muscular  tissue  and  catgut 
or  caoutchouc  therefore  falls  to  the  ground.  Furthermore  even  the 
small  proportion  of  the  structural  elements  of  muscular  tissue  which 
Engelmann  assumes  to  be  subjected  to  heating,  having  been  destroyed 
thereby,  would  have  to  be  decomposed  and  the  products  excreted. 
Muscular  work  should  therefore  consume  muscle-tissue  and  the  nitrog- 
enous excretion  should  increase.  This,  however,  on  a  normal  mixed 
diet,  does  not  occur.  Again,  the  intense  local  heating  which  Engel- 
mann assumes  implies  difficulty  in  the  distribution  and  dissipation  of 
the  heat  which  results  in  muscular  combustion,  yet  the  swift  relaxation 
which  succeeds  normal  muscular  contraction  implies  just  the  reverse. 
A  direct  transformation  of  heat  into  work  through  the  agency  of 
expansion  or  contraction  is  therefore  an  improbable  explanation  of 
muscular  contraction. 

Other  observers  have  sought  to  attribute  the  phenomena  of  mus- 
cular contraction  to  the  Swelling  or  shrinkages  of  semisolid  elements 
through  the  imbibition  or  giving  up  of  water,  as  a  jelly  absorbs  water 
from  or  parts  with  it  to  the  surrounding  medium.  The  known  proc- 
esses of  this  kind  are,  however,  relatively  slow  and  gradual  in  develop- 
ment, whereas  muscular  contraction  and  relaxation  may  in  the  muscles 
of  the  insect's  wing  alternate  no  less  frequently  than  300  times  per 
second. 

The  only  physical  displacements  which  are  capable  at  the  same  time 
of  such  rapid  alternation,  of  the  performance  of  so  much  mechanical 
work  in  a  non-rigid  system,  and  of  transforming  so  large  a  propor- 
tion of  energy  into  mechanical  work  as  a  living  muscle,  are  the  dis- 
placements which  result  from  changes  in  Surface-tension.  These  are 
excessively  rapid  because  the  forces  involved  are  of  great  magnitude 
and  the  frictional  resistances  which  oppose  them  may  be,  under  favor- 
able conditions,  very  small.  The  amount  of  energy  stored  up  in  a 
fluid  surface  is  very  great  and  the  release  of  this  energy  by  chemical 
or  resultant  electrical  changes  affecting  the  tension  of  a  large  surface 
would  suffice  to  permit  the  performance  of  a  large  quantity  of  me- 
chanical work.  The  maximal  attainable  Efficiency  of  a  surface-tension 
engine  as  Brunner  and  Wolf  have  shown,  is  fifty  per  cent.,  i.  e.,  the 
heat  absorbed  in  extending  the  surface  of  water  is  equivalent  to  one- 
half  of  the  mechanical  work  done  in  producing  the  surface-extension. 
This  is  also  the  maximal  efficiency  which  has  ever  been  observed  in  the 
performance  of  muscular  work. 

The  earliest  theory  to  regard  a  muscle  as  a  surface-tension  engine 
was  that  proposed  by  Imbert  who  assumed  that  the  individual  Fibrils 


442    PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

which  are  demonstrable  in  muscular  tissue  are  long  thin  cylinders 
which  are  maintained  in  a  condition  of  elongation  by  passive  stretching. 
Under  the  influence  of  increase  of  surface-tension  at  the  surface  of 
contact  of  these  tubules  and  their  fluid  contents  Imbert  assumes  that 
the  tubules  become  more  spherical  and  therefore  shorter,  the  simul- 
taneous shortening  and  swelling  of  a  number  of  these  elements  leading 
to  the  contraction  of  the  muscle.  According  to  this  hypothesis  the 
work  performed  in  muscular  contraction  is  derived  from  changes  in 
the  surface  energy  of  the  fluid  contained  in  the  tubules.  Bernstein, 
rather  drastically  assuming  certain  magnitudes  for  the  tension  and 
alterations  thereof  at  the  surfaces  of  these  tubules,  inferred  that  if  the 
tubules  consist  of  the  visible  muscle-fibrils  then  the  surface  afforded 
is  not  sufficient  to  account,  on  Imbert's  hypothesis,  for  the  amount  of 
work  performed  in  contraction.  If,  however,  we  suppose  that  the 
fibrils  are  broken  up  into  a  number  of  separate  elements,  for  example 
into  rows  of  ellipsoids  which  become  spherical  when  the  tension  of 
their  surfaces  increases,  then  the  surface  presented  would  be  sufficient 
to  account  for  the  observed  release  of  mechanical  energy.  Now  recent 
investigations  by  Schafer,  McDougall  and  others  on  the  details  of  the 
microscopic  structure  of  muscle,  have  revealed  the  presence  in  the  fibril, 
not  exactly  of  the  structure  imagined  by  Bernstein,  but  one  that  for 
the  purposes  of  Imbert's  hypothesis  is  precisely  equivalent  to  it.  Schafer 
describes  the  contractile  elements  of  the  muscle-fiber  as  fine  columns 
or  Sarcostyles  which  are  divided  into  segments  or  Sarcomeres  by  thin 
transverse  discs,  known  as  Krause's  Membranes.  Each  sarcomere 
contains  a  relatively  opaque  portion,  the  Sarcous  Element,  while  those 
portions  adjacent  to  Krause's  membrane  are  relatively  transparent 
and  seen  to  consist  of  a  fluid  material.  The  sarcous  element  itself  is 
double  and,  if  stretched,  the  two  portions  separate  at  a  line  which  runs 
transversely  across  the  opaque  portion  of  the  sarcomere  (Hensen's  line). 
On  contraction  the  sarcous  elements  become  shorter  and  thicker, 
absorbing  the  fluid  which  constitutes  the  Hyaloplasm  or  intervening 
transparent  area  between  the  sarcous  elements  and  Krause's  mem- 
brane. We  may  therefore  picture  the  muscle-fibril  as  consisting  of  a 
series  of  discs  formed  by  minute  tubules  packed  together  and  com- 
municating with  spaces  separating  the  discs  and  filled  with  fluid  (Fig. 
25).  Evidently  such  a  structure  as  this  conforms  to  every  requirement 
imposed  by  Bernstein  upon  Imbert's  hypothesis,  and  it  is  an  exceedingly 
significant  fact  that  the  details  of  the  structure  which  we  have  out- 
lined become  clearer  and  more  elaborate  as  we  successively  pass  from 
the  relatively  sluggish  and  inert  smooth  muscles,  or  the  striated  mus- 
cles of  amphibia,  to  the  muscles  of  insects  with  their  lightning-like 
rapidity  of  contraction  and  enormous  power  of  performing  work.  This 
fact  alone  prevents  us  from  entertaining  any  doubt  that  this  elaborate 
structure  is  an  essential  part  of  the  muscular  mechanism,  and  the 
salient  characteristic  of  this  structure  is  the  enormous  surface  of 
contact  which  it  brings  about  between  the  fluid  and  the  semisolid 


CONVERSION  OF   CHEMICAL  INTO    MECHANICAL   ENERGY     443 


elements  of  the  tissue.  We  may  therefore  with  considerable  confi- 
dence infer  that  muscular  tissue  is  a  surface-tension  engine  which 
converts  the  energy  released  by  the  combustion  of  carbohydrates  into 
heat  and  mechanical  work. 

Several  mechanisms  are  imaginable  whereby  the  chemical  changes 
which  accompany  muscular  work  might  bring  about  alterations  of 
surface  tension  at  interfaces  within  the  tissue.  The  Heat  of  Combus- 
tion of  carbohydrates  must  of  itself  contribute  to  affect  the  tension  and 
the  changes  of  Electrical  Potential  which  also  accompany  muscular 
contraction  would  likewise,  as  is  shown  by  the  analogy  of  Lipmann's 


FIG.  25. — Wave  of  contraction  passing  over  a  leg-muscle  fiber  of  water-beetle. 

(After  Schafer.) 

capillary  electrometer,  affect  the  tension  of  interfaces  in  the  tissue. 
In  this  connection  one  fact  should  be  very  particularly  noted,  and  that 
is  that  either  of  these  factors,  and  whether  they  are  determinative  or 
not  they  must  contribute  in  some  measure  to  the  outcome,  would  lead, 
not  to  an  increase  of  superficial  tension,  as  imagined  by  Imbert  and 
Bernstein,  but  to  a  decrease.  We  are  therefore  led  to  inquire  whether, 
after  all,  the  alteration  in  form  of  the  sarcous  elements  in  contraction 
may  not  be  due  to  a  decrease  rather  than  to  an  increase  in  interfacial 
tension,  for  otherwise  the  thermal  and  electrical  changes  which  accom- 
pany muscular  contraction  must  actually  diminish  and  inhibit  con- 
traction and  conflict  with  the  main  objective  of  the  whole  process. 


444     PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

A  mechanism  whereby  reduction  of  interfacial  tension  might  bring 
about  the  shortening  of  sarcous  elements  is  depicted  in  the  accom- 
panying figure  (Fig.  26).  If  we  suppose  the  sarcous  element  to  consist 
of  an  elastic  tube  dipping  into  and  filled  by  the  fluid  hyaloplasm,  then 
the  tension  of  this  fluid,  pulling  upon  the  elastic  tubule  will  draw  the 
walls  inward  and  hence  stretch  them  longitudinally.  This  condition  of 
balanced  tensions,  capillary  and  elastic,  may  be  supposed  to  be  the 
normal  resting  state  of  muscle.  If,  now  by  heat,  an  electrical  potential, 
or  other  means,  the  inward  pull  upon  the  tubule  is  released,  this  will 
have  just  the  same  effect  that  internal  pressure  would  have  upon 
an  initially  unstretched  elastic  tube — it  will  expand  and  shorten,  as 
a  hose  expands  and  shortens  when  water  under  a  head  or  pressure  is 
suddenly  injected  into  it.  Thus  the  ends  of  the  sarcous  elements  will 
approach  and,  the  total  capacity  of  each  element  being  increased,  fluid 


Krause's  Membrane 


Sarcous 
Element 


—  Fine  Septa 


*  —  Hansen's  Line 


Krause's  Membrane 
FIG.  26. — Schematic  diagram  of  a  muscle-element. 

from  the  hyaloplasm  will  enter  them.  The  sum  of  the  effects  of  a  multi- 
tude of  such  shortenings  constitutes  the  contraction  of  a  muscle-fibril. 
The  conception  of  a  motile  mechanism  as  a  surface-tension  engine 
may  readily  be  extended  to  include  ameboid  and  ciliary  motion  as 
well  as  the  phenomenon  of  Protoplasmic  Streaming  which  is  so  frequently 
displayed  in  cells  in  which  ameboid  motion  is  constrained  by  viscosity 
or  by  rigid  walls,  as  in  many  plant-cells.  The  genesis  of  movements 
analogous  on  the  one  hand  to  ameboid  motion  and  on  the  other  to 
protoplasmic  streaming  may  be  illustrated  in  a  simple  model  as  follows: 
f  to  a  ten  per  cent,  solution  of  camphor  in  benzole  a  little  dye,  for 
example  Sudan  III  or  Scharlach  R  be  added,  to  render  the  outline  of  a 
drop  visible  against  a  colorless  background,  and  small  drops  of  this  be 
placed  upon  the  surface  of  clean  water  in  a  watch-glass,  very  rapid  and 
energetic  movements  of  the  edges  of  the  drops  may  be  observed  exactly 
similar  in  character  to  those  presented  by  the  surface  of  Ameba. 


CONVERSION   OF   CHEMICAL   INTO    MECHANICAL   ENERGY    445 

Even  processes  similar  in  form  to  Pseudopodia  are  thrown  out  and 
retracted.  These  movements  are  due  to  the  changes  in  interfacial 
tension  caused  by  unequal  diffusion  of  the  camphor  from  the  benzole 
into  the  water.  They  may  be  slowed  by  adding  some  viscous  fluid, 
e.  g.,  olive  oil  to  the  benzol  solution  and  finally,  when  about  an  equal 
volume  of  olive  oil  has  been  added,  we  no  longer  obtain  ameboid 
movements  but,  instead,  we  observe  an  incessant  streaming  movement 
of  the  fluid  within  the  drops,  exactly  resembling  those  seen  within  the 
protoplasm  of  a  plant-cell  such  as  Chara.  If  the  streaming  movements 
are  not  easily  perceived  owing  to  the  transparency  of  the  drop,  the 
addition  of  a  little  finely  powdered  arrowroot  starch  will  render  them 
manifest,  and  impose  a  still  more  close  resemblance  to  the  actual 
appearance  of  streaming  movements  in  protoplasm. 

REFERENCES. 

INFLUENCE  OF  TEMPERATURE  UPON  LIFE  PROCESSES: 

Cohen:     Physical  Chemistry  for  Physicians  and  Biologists,  trans,  by  Fischer,  New 

York,  1903. 

Robertson:     Biol.  Bull.,  1906, 10,  p.  242.     Arch.  Internat.  de  PhysioL,  1908,  6,  p.  388. 
Maxwell:     Jour.  Biol.  Chem.,  1907,  3,  p.  359. 
Lucas:     Jour.  Physiol.,  1908,  37,  p.  112.     The  Conduction  of  the  Nervous  Impulse, 

London,  1917. 

Ganter:     Pfliiger's  Arch  ,  1912,  146,  p.  185. 
Loeb:     The  Mechanistic  Conception  of  Life,  Chicago,  1912.     The  Organism  as  a 

Whole,  New  York,  1916. 

Kanitz:     Tempera tur  und  Lebensvorgange,  Berlin,   1915   (consult   for  literature). 
INFLUENCE  OF  LIGHT  UPON  LIFE  PROCESSES: 

Kober:     Jour.  Biol.  Chem.,  1915,  22,  p.  433. 

Harris  and  Hoyl:     Science,  N.  S.,  1917,  46,  p.  318. 

Loeb:     Forced   Movements,    Tropisms  and  Animal  .Conduct,   Philadelphia,    1918 

(consult  for  literature). 
PHOTOSYNTHESIS  : 

Palladin:     Plant  Physiology,  trans,  by  Livingstone,  Philadelphia,  1918. 
Jost:     Plant  Physiology ,  trans,  by  Gibson,  Oxford,   1907. 
Czapek:     Biochemie  der  Pflanzen.   Jena,   1913. 

Blackman  and  Matthaei:     Proc.  Roy.  Soc.,  London,  1905,  B  76,  p.  402. 
Usher  and  Priestley:     Ibid.,  1905-6,  77,  p.  369;  1906,  78,  p.  318;  1911-12,  84,  p.  101. 
Loeb,  W.:     Zeit.  f.  Elektrochem.,  1905,  11,  p.  745. 
Parkin:     Biochemical  Jour.,  1912,  6,  p.  1. 
MUSCULAR  CONTRACTION  AND  MOTILITY  OF  PROTOPLASM: 
Imbert:     Archives  d.  Physiol.,  5th  ser. ,  1897,  9,  p.  289. 

McDougal:     Jour  of  Anat.  and  Physiol.,  1897,  31,  pp.  410  and  539;  1898,  32,  p.  187. 
Bernstein:     Pfliiger's  Arch.,  1901,  85,  p.  271. 
Robertson:     Trans.  Roy.  Soc.  of  South  Australia,  1905,  29,  p.  1.     Quar.  Jour.  Exp. 

Physiol.,    1909,   2,   p.    303.     Science,    N.   S.,    1912,   36,   p.   446. 
Lillie:     Am.  Jour.  Physiol.,  1908,  22,  p.  75. 
Maxwell   and   Rayleigh:     Article   on    Capillary   Action,    llth    ed.,    Encyclopedia 

Britannica,  5,  p.  256. 


CHAPTER  XIX. 

PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION: 
FERTILIZATION  AND  EARLY  DEVELOPMENT. 

THE  SUBSTITUTION  OF  CHEMICAL  AGENCIES  FOR  NORMAL 
FERTILIZATION. 

IN  the  vast  majority  of  animal  forms  the  stimulus  of  fertilization  by 
a  spermatozoon  of  the  same  or  a  very  closely  related  species  is  essential 
for  the  development  of  the  egg.  The  fact,  however,  that  Partheno- 
genesis, or  development  without  fertilization  may  occur  under  excep- 
tional circumstances  or  in  a  limited  number  of  forms,  shows  that  the 
part  played  by  the  spermatozoon,  in  so  far  as  it  constitutes  the  stimulus 
to  development,  may  be  performed  by  other  agents.  The  discovery 
of  the  exact  nature  of  agents  capable  of  giving  rise  to  development 
of  the  egg  was  essential  to  the  understanding  of  the  phenomenon  of 
Fertilization,  for  the  spermatozoon,  besides  affording  to  the  egg  the 
initiatory  impulse  to  development  also  acts  as  a  bearer  of  hereditary 
factors  and  is,  moreover,  itself  a  living  and  a  motile  organism  so  that  a 
great  complexity  of  materials  and  factors  gain  entry  into  the  egg  with 
the  introduction  of  the  spermatozoon,  and  the  disentanglement  of 
these  numerous  variables  was  impossible  until  a  clue  to  their  nature 
had  been  obtained  by  means  of  experiments  in  which  the  single  func- 
tion of  fertilization  was  imitated  by  physicochemical  means. 

The  solution  of  this  problem  we  owe  to  the  investigations  of  J.  Loeb 
who  followed  up  the  observation  of  T.  H.  Morgan  and  others  that 
unfertilized  eggs  of  various  marine  organisms  may  occasionally  begin 
to  segment  without  fertilization  in  sea-water,  but  that  such  eggs 
invariably  die  after  a  few  divisions.  In  seeking  to  ascertain  the 
origin  of  this  abnormal  phenomenon  Loeb  found  that  in  the  eggs 
of  a  sea-urchin,  Arbacia,  development  could  be  induced  by  merely 
exposing  them  for  a  period  to  slightly  Hypertonic  Sea-water  and 
then  returning  them  to  normal  sea-water.  The  means  employed 
to  render  the  sea-water  hypertonic  was,  within  certain  limits,  imma- 
terial. Thus  the  Osmotic  Pressure  might  be  raised  by  spontaneous 
evaporation,  or  by  the  addition  of  one  part  by  volume  of  1\  normal 
sodium  chloride  solution  to  nine  parts  by  volume  of  sea-water,  or  yet 
again  Cane-sugar  or  Urea  or  some  other  physiologically  inert  substance 
might  be  employed  for  this  purpose  and  with  like  success.  It  was 
even  found  possible  to  cause  development  of  the  eggs  by  immersing 
them  in  a  pure  cane-sugar  solution  only  slightly  exceeding  sea-water 


SUBSTITUTION  OF  AGENCIES  FOR  FERTILIZATION       447 

in  its  osmotic  pressure.  The  increase  of  osmotic  pressure  required  is 
not  great.  If  Sodium  Chloride  be  employed  an  increase  of  forty  per 
cent,  in  the  osmotic  pressure  of  the  sea- water  suffices  to  initiate  develop- 
ment after  an  exposure  of  two  hours.  If  sugar  or  urea  be  employed 
even  a  slighter  increase  of  osmotic  pressure  suffices  to  bring  about  a 
like  effect,  because  these  substances  penetrate  the  egg  with  greater 
difficulty  than  the  inorganic  salts  and  hence  exert  a  greater  osmotic 
tension  on  the  external  surface  of  the  egg.  The  requisite  concen- 
tration of  the  medium  varies,  however,  with  the  duration  of  the  expos- 
ure, a  weaker  concentration  being  effective  after  a  longer  exposure. 
This  is  shown  by  the  following  experiment:  To  50  c.c.  portions  of 
artificial  sea- water  (Van  t'Hoff's  Solution)  rendered  favorably  alkaline 
by  the  addition  of  2  c.c.  of  tenth  normal  sodium  hydroxide  were  added 
0, 2, 4,  8  and  16  c.c.  of  2|  normal  potassium  chloride  solution.  Unfertil- 
ized eggs  of  a  Pacific  Ocean  sea-urchin  (Strongylocentrotus  purpuratus) 
were  divided  between  these  five  solutions  and  samples  removed  after 
varying  periods  of  exposure  and  placed  in  normal  sea-water.  The 
following  were  the  results  obtained: 

Increase  in  the  osmotic  pressure  of  the  medium. 
Period  of  exposure, 


minutes.  0  per  cent.       16  per  cent.       30  per  cent.        55  per  cent.         87  per  cent. 

45     ....      No  larvae       No  larvae       No  larvae         No  larvae          Numerous 

larvae. 
64     ....  "  "  "  Numerous 

larvae 
89     .      .    ..      .'  "  Numerous 

larvae 

116     ..'..;. 
114     .      . 

The  fertilization  which  resulted  from  this  procedure  failed,  however, 
to  furnish  a  perfectly  faithful  imitation  of  the  phenomenon  of  natural 
fertilization.  It  is  true  that  the  eggs  frequently  developed  into  free- 
swimming  larvae,  but  the  larvae  produced  in  this  manner  were  sickly 
and  abnormal  and  did  not  survive  very  long.  The  percentage  of  eggs 
which  developed  into  larvae  was  variable  and  in  some  species,  partic- 
ularly in  the  sea-urchin  Strongylocentrotus  franciscanus,  few  if  any  of 
the  eggs  could  be  induced  to  develop  by  this  procedure.  The  larvae  in 
all  cases  behaved  abnormally;  they  did  not  rise  to  the  top  of  the  water 
and  swim  there  as  normal  larvae  do,  but  swam  instead  at  the  bottom  of 
the  vessel  containing  them,  and  finally,  the  most  marked  peculiarity 
of  all  was  the  failure  of  the  eggs  to  form  a  Fertilization-membrane. 

If  the  eggs  of  a  mature  female  sea-urchin  be  removed  from  the 
ovaries  by  shaking  them  out  in  sea- water  and  are  then  mixed  with 
sperm  similarly  procured  from  the  spermaries  of  a  male,  the  sperma- 
tozoa will  immediately  be  seen  clustering  around  the  eggs,  presenting 
the  appearance  of  striving  to  enter  them.  Within  a  very  brief  period, 
under  normal  conditions,  a  spermatozoon  will  succeed  in  effecting  an 
entry,  and  this  event  is  at  once  indicated  by  the  appearance  upon  the 


448     PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

surface  of  the  egg  of  a  number  of  irregular  clear  blister-like  protuber- 
ances which  rapidly  increase  in  number  and  extent,  finally  covering 
the  surface  of  the  egg  with  a  clear  hyaline  layer,  which  is  designated 
the  fertilization-membrane  (Fig.  27).  This  was  lacking  in  the  artifi- 
cially fertilized  egg. 

The  imperfect  character  of  the  imitation  of  fertilization  which  was 
thus  achieved  led  Loeb  to  form  the  supposition  that  the  osmotic  method 
of  inducing  fertilization  only  accomplished  a  part  of  the  effects  ini- 
tiated by  the  spermatozoon,  which  he  inferred  carried  into  the  egg 
agencies  not  only  capable  of  starting  the  processes  initiated  by  the 
hypertonic  sea-water  but  also  processes  which  the  osmotic  method 
did  not  suffice  to  initiate.  This  supposition  was  confirmed  by  the 
discovery  of  a  series  of  agents  capable  of  inducing  Membrane -forma- 
tion in  the  sea-urchin  egg. 


1.  2. 

FIG.  27. — 1,  unfertilized  egg  of  the  Sea-urchin  (Strongylocentrotus  purpuratus)  sur- 
rounded by  spermatozoa;  2,  the  same  egg  about  two  minutes  later,  after  the  entrance 
of  the  spermatozoon  and  the  formation  of  the  fertilization-membrane.  (After  Loeb.) 

It  was  found  that  if  mature  sea-urchin  eggs  were  introduced  for  a  few 
minutes  into  sea-water  to  which  a  small  proportion  of  a  certain  sample 
of  Ethyl  Acetate  had  been  added,  and  then  returned  to  normal  sea-water, 
all  of  the  eggs  promptly  formed  a  fertilization-membrane  differing  in 
no  perceptible  degree  from  the  membranes  formed  in  normal  fertili- 
zation. Other  esters  failed  to  yield  any  comparable  result,  and  an 
examination  of  the  ethyl  acetate  employed  in  the  original  experiment 
showed  that  it  had  undergone  hydrolysis  and  contained  free  ethyl 
alcohol  and  acetic  acid.  This  led  to  an  investigation  of  the  behavior 
of  the  eggs  in  sea- water  containing  added  alcohols  and  acids  and  to  the 
discovery  that  the  effect  originally  obtained  with  impure  ethyl  acetate 
was  due  to  the  Acetic  Acid  which  it  contained.  It  was  found  that  all 
of  the  monobasic  fatty  acids  which  are  soluble  in  sea-water,  namely 
formic  acid,  acetic  acid,  propionic  acid,  butyric  acid,  valerianic  acid 
and  so  forth,  will  induce  membrane  formation  in  100  per  cent,  of 
mature  eggs  if  they  are  exposed  for  a  brief  period  to  the  action  of  the 
sea- water  containing  the  fatty  acid.  The  formation  of  the  membrane 


SUBSTITUTION  OF  AGENCIES  FOR  FERTILIZATION       449 

does  not  occur  until  after  the  restoration  of  the  egg  to  the  normal 
sea-water. 

The  eggs  which  have  been  treated  in  this  manner  may  undergo  a  few 
divisions  but  they  very  rapidly  die,  more  rapidly,  in  fact,  than  unfertil- 
ized eggs  exposed  to  similar  conditions  of  temperature,  etc.  The 
processes  thus  initiated  therefore,  still  afford  an  incomplete  analogy 
to  natural  fertilization.  It  was  found,  however,  that  by  a  combination 
of  these  two  processes,  membrane-formation  and  osmotic  treatment, 
which  are  separately  incomplete,  a  perfect  imitation  of  fertilization  is 
procured  and  a  high  percentage  of  the  eggs,  usually  100  per  cent.,  can 
be  induced  to  develop  and  produce  normal  larvae. 

The  precise  details  of  time,  of  exposure,  concentration  and  so  forth, 
in  Loeb's  improved  method  of  Artificial  Parthenogenesis  necessarily 
vary  slightly  with  the  temperature,  reaction  of  the  sea-water  and 
species  of  Echinoderm  employed.  The  following  are,  however,  the 
details  of  the  method  as  utilized  for  the  fertilization  of  the  Pacific 
sea-urchin,  Strongylocentrotus  pur  pur  aim  at  a  temperature  in  the 
neighborhood  of  15°  C.  The  eggs,  after  extraction  from  the  ovaries 
and  rinsing  in  filtered1  sea-water  are  immersed  in  a  mixture  of  50  c.c. 
of  sea-water  and  2.8  c.c.  of  tenth  normal  Butyric  Acid  solution,  and 
the  mixture  is  gently  agitated  to  prevent  the  eggs,  which  become  sticky, 
from  adhering  to  the  bottom  of  the  vessel.  After  about  two  minutes  the 
eggs  are  collected  by  gentle  rotation  of  the  shallow  flat-bottomed 
vessel  and  transferred  by  means  of  a  pipette  to  normal  sea-water.  If 
the  exposure  has  been  rightly  chosen  it  will  be  found  that  the  eggs 
almost  immediately  form  membranes.  After  allowing  them  to  remain 
for  some  fifteen  to  twenty  minutes  in  the  normal  sea-water  they  are 
again  collected  in  the  manner  described  and  transferred  to  Hypertonic 
Sea-water,  prepared  by  adding  8  c.c.  of  2J  molecular  sodium  chloride 
solution  to  50  c.c.  of  sea- water.  They  are  exposed  to  this  addition  for 
a  period  varying  from  fifteen  to  sixty  minutes,  the  optimal  exposure 
varying  somewhat  with  the  eggs  from  different  females.  The  eggs  are 
now  returned  to  normal  sea-water.  Within  about  one  hour  the  first 
cell-division  will  be  observed  to  have  occurred,  at  the  end  of  forty- 
eight  hours  swimming  gastrulse  will  have  been  produced,  and  about 
two  days  later  plutei  with  well-developed  skeletons. 

Artificial  fertilization  has  been  extended  to  a  variety  of  forms  other 
than  the  Echinoderms.  In  a  number  of  Annelids  development  may 
be  induced  by  preliminary  treatment  with  a  cytolytic  agent  followed 
by  treatment  with  hypertonic  sea-water.  As  a  rule,  however,  in  this 
group  the  fatty  acids  do  not  constitute  sufficiently  potent  cytolytic 
agents,  and  saponins  or,  better  still,  mammalian  blood  serum  must  be 
employed.  Among  the  Molluscs  simple  treatment  with  hypertonic 
sea- water  frequently  suffices,  especially  if  it  be  rendered  slightly  hyper- 

1  The  sea- water  must   be   filtered  to  remove  spermatozoa  which  may  possibly  be 
suspended  in  it. 
29 


450     PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

alkaline,  the  alkali  playing  the  part  of  the  cytolytic  or  membrane- 
forming  agent.  In  the  eggs  of  frogs  simple  puncture  with  a  fine  needle 
suffices  to  induce  parthenogenetic  development,  for  what  reason  is  not 
at  present  clearly  understood,  although  we  may  fairly  infer  that  it 
arises  from  the  incidental  admixture  of  certain  constituents  of  the  eggs 
which  are  normally  separated  from  one  another.  Artificial  partheno- 
genesis has  also  been  induced  in  the  eggs  of  plants  (Fucus)  by  treating 
them  with  butyric  acid,  followed  by  hypertonic  sea-water. 

The  eggs  of  all  forms  which  have  been  made  to  undergo  develop- 
ment by  artificial  means  yield  normal  embryos  and  their  development 
differs  in  no  wise  from  that  of  normally  fertilized  animals.  The  rearing 
of  marine  animals  is  an  excessively  laborious  task,  but  Delage  has  had 
the  courage  to  undertake  it  in  the  case  of  artificially  fertilized  sea- 
urchins  and  succeeded  in  maintaining  them  until  sexual  maturity. 
In  the  case  of  the  frog  several  specimens  arising  from  artificially 
fertilized  eggs  have  been  brought  to  sexual  maturity  by  Loeb  and 
Bancroft  (see  Fig.  28). 


FIG.  28. — A  parthenogenetic  frog.     (After  Loeb.) 

THE  NATURE  OF  THE  AGENTS  WHICH  FORM  FERTILIZATION- 
MEMBRANES. 

The  monobasic  acids  of  the  fatty  series  are  all  capable  of  producing, 
as  we  have  seen,  the  formation  of  membranes  in  the  sea-urchin  egg 
provided  they  are  soluble  in  sea-water.  Now  this  action  might  con- 
ceivably be  due  to  the  dissociation  of  Hydrogen  Ions  by  the  acids,  or 
it  might  be  due,  on  the  other  hand,  to  the  anion  or  the  undissociated 
molecule  of  the  acid.  The  latter  is  the  correct  alternative,  for  although 
the  highly  dissociated  mineral  acids  will  induce  Membrane-formation 
in  a  limited  percentage  of  eggs,  the  requisite  concentration  of  these 


NATURE  OF  AGENTS  WHICH  FORM  MEMBRANES          451 

acids  is  far  greater  than  it  is  in  the  case  of  the  fatty  acids.  In  fact  one- 
thousandth  normal  Butyric  Acid  is  more  efficient  in  inducing  membrane- 
formation  than  twelfth  normal  Hydrochloric  Acid ;  yet  hydrochloric  acid 
of  even  this  concentration  does  not  injure  the  eggs  in  the  periods  of 
exposure  requisite,  because  normal  fertilization  by  sperm  can  still 
occur  in  100  per  cent,  of  the  eggs  treated  in  this  manner,  and  weaker 
concentrations  of  hydrochloric  acid,  which  are  usually  ineffective  in 
causing  membrane-formation,  are,  of  course,  even  less  toxic.  A  very 
convincing  experiment  devised  by  Loeb  to  illustrate  this  point  consists 
in  adding  a  little  Sodium  Butyrate  to  a  solution  of  hydrochloric  acid 
in  sea-water  which  is  otherwise  incapable  of  causing  membrane-for- 
mation. The  mixture  immediately  becomes  an  effective  membrane- 
forming  agent,  although  its  acidity,  if  anything,  has  been  reduced.  The 
introduction  of  the  sodium  butyrate  leads  to  an  interaction  with  the 
hydrochloric  acid,  setting  free  a  little  butyric  acid  which  accomplishes 
the  initial  stage  of  fertilization. 

It  had  been  observed  in  1887  by  O.  and  R.  Hertwig.that  if  sea- 
urchin  eggs  be  immersed  in  sea-water  saturated  with  Chloroform — 
and  only  a  trace  of  this  substance  will  dissolve  in  sea- water — fertili- 
zation-membranes are  formed.  It  had  also  been  found  by  Herbst 
that  Benzene,  Toluene  and  Creosote  have  a  similar  action.  In  all  these 
cases,  however,  the  membrane-formation  was  found  to  be  rapidly  suc- 
ceeded by  Cytolysis  and  the  disintegration  of  the  eggs,  so  that  develop- 
ment, of  course,  did  not  occur.  Loeb  found  however,  that  if  the  eggs 
be  exposed  only  for  very  brief  periods  to  these  solutions  and  then 
transferred  to  normal  sea-water  a  percentage  of  the  eggs  will  form 
membranes  without  cytolysis  and  may  subsequently  be  induced  to 
develop  by  treatment  with  hypertonic  sea-water.  It  is  a  curious,  and 
as  yet  unexplained  fact  that  whereas  eggs  immersed  in  butyric-acid 
sea-water  do  not  form  fertilization-membranes  until  they  are  trans- 
ferred to  normal  sea-water,  eggs  treated  with  sea-water  containing 
benzene  or  amylene  form  membranes  before  they  are  removed  from 
the  mixture. 

The  various  reagents  which  were  thus  found  effective  in  inducing 
membrane-formation  have  this  in  common,  that  they  are  all  fat- 
solvents  or  highly  soluble  in  fats  and  furthermore  they  are  all  in  greater 
or  less  degree  Hemolytic  Agents,  that  is,  substances  which  are  capable  • 
of  dissolving  red  blood-corpuscles.  This  fact  drew  attention  to  the 
possibility  that  other  hemolytic  agents  might  be  capable  of  exerting 
a  like  effect  upon  the  unfertilized  egg  of  the  sea-urchin. 

According  to  Koeppe,  besides  heat  and  alternating  electric  currents 
there  are  five  distinct  groups  of  chemical  agents  which  are  distinguished 
by  their  power  of  inducing  hemolysis  of  red  blood-corpuscles  or,  more 
generally,  Cytolysis  of  all  types  of  living  cells.  These  are:  1.  Certain 
specific  substances,  for  example  the  series  of  glucosides  comprising 
the  Saponins  and  Solanins,  or  the  Bile-salts.  2.  A  series  of  Fat-sol- 
vents such  as  benzene,  ether  or  alcohol.  3.  Distilled  water,  4.  Hydro- 


452    PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

gen  ions  and  5.  Hydroxyl  ions.    Successive  experiments  have  shown 
that  each  of  these  groups  of  reagents  may,  by  appropriate  manipula- 


6.  7. 

FIG.  29. — Formation  of  fertilization-membrane  and  cytolysis  of  the  sea-urchin  egg 
on  treatment  with  saponin.  1,  appearance  of  egg  when  first  exposed  to  saponin  solu- 
tion at  9.07  A.M.;  2,  3,  and  4,  formation  of  fertilization-membrane.  The  stage  of  com- 
plete membrane-formation  as  depicted  in  4  was  reached  at  9.15  A.M.  (if  at  this  stage 
the  egg  is  withdrawn  from  the  influence  of  the  saponin  it  can  develop).  Cytolysis,  5, 
began  at  9.20  A.M.;  6  and  7,  advanced  stages  of  cytolysis  induced  by  saponin.  (After 
Loeb,) 

tion,  be  made  to  cause  membrane-formation  in  the  unfertilized  sea- 
urchin  egg.  The  Saponins  especially  are  extraordinarily  efficient  in 
inducing  this  phenomenon,  membranes  being  formed  in  100  per  cent. 


NATURE  OF  AGENTS  WHICH  FORM  MEMBRANES         453 

of  eggs  by  a  dilution  of  one  in  a  hundred  thousand  after  an  exposure  of 
from  30  to  60  minutes;  these  membranes  are  formed  in  the  solution 
itself  without  transference  to  normal  sea-water.  If,  however,  the 
action  of  the  saponin  solution  upon  the  egg  be  permitted  to  continue, 
membrane-formation  is  rapidly  succeeded  by  cytolysis  and  the  egg 
disintegrates.  This  is  illustrated  in  the  preceding  figure  (Fig.  29), 
showing  the  successive  effects  of  a  saponin  solution  (8  drops  of  J  per 
cent,  saponin  to  five  c.c.  of  sea-water)  upon  the  unfertilized  eggs  of 
Strongylocentrotus.  It  is  perfectly  clear  from  these  results  that  the 
formation  of  the  fertilization  membrane  represents  an  initial  stage  of 
cytolysis.  If,  however,  the  eggs  be  removed  from  the  saponin  solution 
before  manifest  cytolysis  has  occurred,  washed  in  sea-water  a  number 
of  times  to  remove  the  last  traces  of  saponin,  and  then  treated  with 
hypertonic  sea-water,  normal  development  occurs  and  a  large  propor- 
tion of  the  eggs  develop  into  larvae.  Precisely  the  same  results  are 
obtained  with  Bile-salts. 

There  remains,  however,  another  class  of  cytolytic  agent  which  has 
yet  to  be  considered  in  this  connection,  namely  the  tissue-fluids  of 
unrelated  species  of  animals.  The  Blood  of  the  mammalia  contains 
cytolytic  substances  which  hemolyze  and  destroy  foreign  corpuscles 
and  cells,  but  not  those  of  the  same  species.  This  cytolytic  power  of 
blood  and  other  tissue-fluids  is  greatly  enhanced  by  previous  immuni- 
zation with  the  foreign  cells,  but  within  the  body  at  least  it  is  exercised 
without  previous  immunization.  It  was  found  by  Loeb  that  this  class 
of  cytolytic  agents  is  also  capable  of  causing  membrane-formation 
in  the  sea-urchin  egg.  Not  every  unrelated  species  of  animal,  however, 
would  furnish  a  tissue-fluid  or  extract  capable  of  causing  membrane- 
formation,  in  fact  only  a  limited  number  of  forms  were  found  to  do  so. 
Loeb  considers  that  this  is  due  to  the  variable  permeability  of  the  eggs 
for  different  lysins,  and  the  permeability  of  the  eggs  for  a  particular 
lysin  also  seems  to  vary  somewhat  in  the  eggs  of  different  females. 

The  first  cytolytic  agent  of  this  kind  to  be  discovered  was  that 
contained  in  the  blood  of  certain  marine  worms,  namely  Dendrostoma, 
which  calls  forth  membrane-formation  in  the  sea-urchin  egg  even  if  it  is 
diluted  a  thousand  times  or  more  with  sea-water.  Later  investigations 
have  shown  that  a  number  of  other  invertebrates  yield  tissue-fluids 
or  extracts  which  will  cause  membrane-formation.  Most  interesting 
results  were,  however,  obtained  by  Loeb  with  the  blood  of  mammalia 
or  birds. 

The  blood-sera  of  mammals  (oxen,  sheep  or  pigs)  which  have  been 
rendered  isotonic  with  sea-water  by  the  addition  of  sodium  chloride 
will  induce  membrane-formation  in  sea-urchin  eggs,  but  not  invariably 
nor  in  all  of  the  eggs  derived  from  different  females.  Eggs  from  one  and 
the  same  female  will  form  membranes  in  some  samples  of  blood  and  not 
in  others,  and  again,  one  and  the  same  sample  of  blood  will  form 
membranes  in  the  eggs  of  some  females  but  not  in  others.  As  in  the 
case  of  the  saponins  the  membranes  are  formed  in  the  solution  itself, 


454    PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

and  removal  to  normal  sea-water  is  not  essential,  but,  unlike  the  mem- 
brane-formation by  saponins  it  is  not  followed,  in  undiluted  blood  at 
least,  by  subsequent  cytolysis.  As  we  shall  see,  however,  dilution  of 
the  blood  by  sea-water  enables  the  cytolytic  effect  to  appear,  complet- 
ing the  analogy  to  the  action  of  the  saponins.  As  in  all  the  instances 
previously  considered,  membrane-formation  is  succeeded  by  one  or 
two  cell-divisions  and  then  by  death  and  disintegration  of  the  eggs, 
unless  they  are  treated  with  hypertonic  sea-water  which  enables  them 
to  develop  and  give  rise  to  normal  embryos. 

Loeb  sought  for  the  origin  of  the  differing  action  of  the  various  sera 
by  endeavoring  to  modify  it,  and  he  found  that  preliminary  heating 
of  the  sera  greatly  enhanced  their  ability  to  induce  fertilization.  Pre- 
liminary treatment  of  the  eggs,  however,  was  found  to  be  even  more 
effective.  This  treatment  or  "sensitization"  of  the  eggs  consists  in 
exposing  them  for  a  brief  period  to  an  isotonic  solution  of  a  chloride  of 
an  Alkaline  Earth,  calcium,  strontium  or  barium.  Of  the  three,  however, 
strontium  is  much  the  most  effective;  the  efficiency  of  barium  might 
possibly  exceed  even  that  of  strontium  if  it  were  not  for  the  fact  that 
barium  is  also  exceedingly  toxic  for  the  eggs,  while  strontium  is  almost 
harmless.  After  exposure  to  the  Strontium  Chloride  and  subsequent 
transference  to  isotonic  blood-serum,  membranes  are  formed  in  nearly 
every  case  upon  100  per  cent,  of  the  eggs.  It  has  been  shown  by  A.  R. 
Moore  that  this  sensitization  by  strontium  is  due,  not  to  any  irreversible 
change  induced  in  the  egg  itself  by  strontium,  but  more  probably  to 
the  actual  presence  of  the  strontium  within  the  egg,  for  if  the  eggs 
after  exposure  to  the  strontium  chloride  solution  be  washed  free  of  the 
solution  by  two  or  three  changes  of  sea-water,  their  sensitiveness  to 
blood-sera  is  lost. 

The  membrane-forming  substance  in  blood-serum  was  found  by 
Loeb  to  be  remarkably  resistant  to  heat.  Exposure  of  ox-serum  to  a 
temperature  of  73°  for  half  an  hour,  which  leads  to  coagulation  of  the 
serum-proteins,  does  not  destroy  its  fertilizing  power.  Heating  the 
serum  to  100°  does,  however,  destroy  the  active  substance.  The  blood 
of  Dendrostoma  still  retains  a  proportion  of  its  membrane-forming 
power,  even  after  having  been  heated  rapidly  to  boiling  point;  more 
prolonged  boiling  (2  to  3  minutes),  however,  destroys  its  activity. 
The  thermostabile  character  of  the  active  substance  at  once  distin- 
guishes it  from  the  Alexin  or  bactericidal  substance  which  is  present  in 
mammalian  blood-seia,  for  this  is  inactivated  by  heating  to  56°. 

The  active  substance  in  mammalian  sera  is  not  extracted  by  shaking 
the  serum  with  Ether.  If  several  volumes  of  Acetone  are  added  to 
the  serum,  the  precipitate  which  results,  after  drying,  powdering  and 
resolution  in  sea-water,  retains  the  power  of  inducing  membrane- 
formation.  The  substance  is  therefore  precipitated  by  acetone. 

A  very  minute  and  intensely  active  fraction  may  be  prepared  from 
mammalian  blood-sera  by  the  following  procedure.  One  hundred  c.c. 
of  ten  per  cent.  Barium  Chloride  solution  are  added,  with  constant 


NATURE  OF  AGENTS  WHICH  FORM  MEMBRANES         455 

stirring,  to  each  liter  of  serum.  This  mixture  is  kept  in  a  cool  place 
for  forty-eight  hours,  when  the  supernatant  fluid  can  be  decanted.  The 
residue  is  washed  several  times  with  large  volmes  of  2  per  cent,  barium 
chloride  solution  to  remove  all  traces  of  serum.  The  precipitate  is  now 
stirred  up  with  tenth-normal  hydrochloric  acid,  warmed  to  45°  C. 
using  50  c.c.  for  the  precipitate  from  each  liter  of  serum.  After  stirring 
for  an  hour  or  more  the  mixture  is  centrifuged  and  to  the  clear  fluid 
thus  obtained  an  equal  volume  of  tenth-normal  sulphuric  acid  is 
added.  This  solution  is  allowed  to  stand  at  40°  C.  for  twenty-four  hours 
and  then  thoroughly  centrifuged  to  remove  barium  sulphate.  To  the 
clear  fluid  are  added  four  or  five  volumes  of  acetone  and  the  mixture 
is  cooled  for  eight  hours  or  more,  at  the  end  of  which  time  the  white 
flocculent  precipitate  has  settled.  It  can  then  be  collected  on  a  hard- 
ened filter,  washed  with  ether  and  dried  over  sulphuric  acid.  This 
preparation,  which  has  been  designated  Oocytin  has  been  carefully 
examined  by  G.  W.  Clark,  who  finds  that  successive  samples  differ  in 
elementary  composition,  showing  clearly  that  it  is  a  mixture  of  two  or 
more  substances.  It  gives  all  the  protein  reactions  but  also  yields  on 
hydrolysis  notable  quantities  of  Hypoxanthine  and  a  Pentose,  but  only 
a  trace  of  phosphoric  acid.  These  products  correspond  to  those  which 
would  be  yielded  by  the  Nucleosides  or  glucosidal  fractions  derivable 
from  the  nucleic  acids  by  partial  hydrolysis.  The  presence  of  appre- 
ciable amounts  of  this  glucoside  in  the  preparation  is  of  peculiar 
significance  when  the  glucosidal  structure  of  the  Saponins,  which  are 
similarly  potent  in  inducing  membrane-formation,  is  borne  in  mind. 

The  membrane-forming  power  of  oocytin  acting  upon  eggs  sensi- 
tized by  strontium  chloride  is  very  great,  comparable  in  fact  with  that 
of  the  saponins.  It  will  induce  membrane-formation  at  a  dilution  of 
one  in  five  hundred  thousand.  The  sensitizing  effect  of  strontium  is 
clearly  seen  to  lie  in  the  fact  that  it  precipitates  the  oocytin  and  so 
concentrates  it  within  or  upon  the  surface  of  the  egg.  In  concentrated 
solutions  eggs  which  have  been  freshly  transferred  from  strontium 
chloride  solution  collect  a  dense  precipitate  at  their  periphery  which 
may  often  mechanically  prevent  or  delay  the  formation  of  the  fertili- 
zation-membrane. That  the  activity  of  the  preparation  is  not  due  to 
contamination  by  barium  or  other  inorganic  substances  is  shown  by  the 
fact  that  it  is  inactivated  by  heating  for  a  few  minutes  to  80°  Q. ;  the 
temperature  at  which  the  activity  of  blood-serum  itself  is  destroyed. 

From  the  spermatozoa  of  the  sea-urchin,  cytolyzed  by  distilled  water, 
a  similar  fraction  may  be  prepared  having  analogous  potency  in 
inducing  membrane-formation  in  eggs  which  have  been  sensitized  by 
immersion  in  strontium  chloride  solution.  This  material  has  also  been 
found  by  Clark  to  yield  notable  amounts  of  a  purine  base  and  a  pen- 
tose  on  hydrolysis.  It  would  appear  very  probable  therefore  that 
membrane-formation  in  natural  fertilization  is  brought  about  by  the 
introduction  into  the  egg,  within  the  body  of  the  spermatozoon,  of  a 
glucosidal  cytolytic  agent,  which  is  related  to  the  nucleosides. 


456     PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

The  action  of  oocytin  upon  the  sea-urchin  egg  differs  from  that  of 
isotonic  mammalian  blood  serum  in  two  respects;  firstly  in  the  fact 
that  prolonged  exposure  of  the  eggs  to  oocytin  solutions  causes  cytoly- 
sis  and  secondly  in  the  fact  that  it  is  potent  at  a  great  number  of  differ- 
ent dilutions  whereas  the  potency  of  isotonic  serum  to  induce  mem- 
brane-formation disappears  upon  dilution  of  the  serum  to  one-half,  or 
at  all  events  one-fourth,  by  the  addition  of  sea-water.  This  differ- 
ence in  behavior  is  apparent,  however,  and  not  real.  It  is  due  to  the 
inhibiting  action  of  the  proteins  which  are  also  present  in  the  serum. 

If  isotonic  blood-serum  be  diluted  by  successive  additions  of  sea- 
water  the  membrane-forming  power  is  at  first  weakened  and  then 
disappears,  but  upon  further  dilution  it  reappears  and  then  is  retained 
to  very  high  dilutions.  The  following  are  illustrative  experiments: 

OX-SERUM  SAMPLE  III. 
Eggs  sensitized  by  four  minutes'  immersion  in  f  m.  SrCl2 

Dilution  of  the  Per  cent,  of  membrane  Per  cent,  of  membrane 

isotonic  formed  in  fifteen  formed  in  fifty 

serum.  minutes.  minutes. 

1 100  100 

1/2 5  50 

1/4 0  0 

1/8  .  ' Not  observed  44 

1/16 100 

OX-SERUM  SAMPLE  V. 
Eggs  sensitized  by  four  minutes'  immersion  in  f  m.  SrCl2. 

Dilution  of  the  Per  cent,  of  membrane  Per  cent,  of  membrane 

isotonic  formed  in  fifteen  formed  in  ninety 

serum.  minutes.  minutes. 

1 68  100  (none  cytolyzed) 

1/2 80  86  (none  cytolyzed) 

1/4 0  0  (none  cytolyzed) 

1/8 0  16  (   1  per  cent,  cytolyzed) 

1/16 1  72  (10  per  cent,  cytolyzed) 

It  is  evident  that  when  the  membrane-forming  power  is  regained  in 
the  higher  dilutions  the  power  of  inducing  cytolysis  is  also  acquired, 
so  that  the  action  of  the  blood-serum  now  resembles  that  of  saponin 
or  oocytin  in  every  respect.  The  failure  of  cytolysis  to  appear  in 
undiluted  serum  is  due  to  the  inhibiting  effect  of  the  high  concentration 
of  Protein  which  it  contains,  and  even  membrane-formation  may  be 
inhibited  if  the  concentration  of  serum-proteins  is  too  high  or  if  addi- 
tional protein  be  dissolved  in  the  serum.  Such  sera  will  nevertheless 
induce  membrane-formation  and  cytolysis  if  they  are  diluted,  the  inhib- 
iting effect  of  the  proteins  becoming  negligible  at  a  dilution  of  one  in 
sixteen  or  one  in  thirty-two. 

Even  membrane-formation  by  Butyric  Acid  or  by  spermatozoa  may 


NATURE  OF  AGENTS  WHICH  FORM  MEMBRANES         457 

be  inhibited  by  the  addition  of  proteins  to  the  sea- water.  The  following 
table  shows  the  relative  efficiency  of  various  proteins  in  inhibiting 
membrane-formation : 

Highest  observed  Lowest  observed 
concentration  which  concentration  which 
permits  membrane-  prevents  membrane- 
formation  by  formation  by 
Protein.                                                  butyric  acid.  butyric  acid. 

Mixed  serum  proteins 3.7  7.4 

Gelatin ..      .      .      .  1.0  2.0 

"Insoluble"  serum-globulin  ....  0.3  0.6 

Casein ,      ...  0.25  0.5 

Ovomucoid.      .      ...      .      .      .      .      .  0.125  0.25 

It  is  a  very  striking  fact  that  the  order  of  effectiveness  of  these 
proteins  in  preventing  the  formation,  of  membranes  is  the  reverse 
order  of  their  ability  to  pass  through  a  porcelain  filter.  It  has  been 
suggested  by  Loeb  and  von  Knaffle-Lenz  that  the  formation  of  the  fer- 
tilization-membrane is  accompanied  by  the  entry  of  water  into  the  egg. 
This  is  prevented  or  delayed  by  the  presence  of  colloids  in  the  sur- 
rounding medium  because  they  cannot  penetrate  the  egg  and  hence 
exert  an  osmotic  pressure  tending  to  withdraw  water  from  the  egg. 
For  similar  reasons  cytolysis  is  also  inhibited  and  it  has  also  been 
stated  by  B.  Moore  that  the  action  of  Hemolytic  Agents  in  liquefying 
blood-corpuscles  is  similarly  inhibited  by  proteins. 

The  normal  concentration  of  protein  in  blood-serum  lies  between 
7  and  8  per  cent,  and  it  will  be  seen  that  this  lies  in  the  margin  of  the 
concentration  which  inhibits  membrane-formation  by  butyric  acid 
(and  also  by  sperm).  Hence  if  the  oocytin  content  of  a  sample  of 
serum  be  low,  or  the  concentration  of  serum-proteins  a  little  above  the 
average,  it  will  fail  to  cause  membrane-formation  even  in  sensitized 
eggs.  Heating  the  serum  permits  membrane-formation  to  occur 
because  it  results  in  coagulating  and  removing  the  proteins,  and  dilu- 
tion achieves  the  same  result  in  a  different  way.  At  first,  however, 
the  effect  of  dilution  in  reducing  the  membrane-forming  power  of  the 
serum  more  than  compensates  for  the  diminished  inhibition  by  the 
proteins,  so  that  dilution  of  serum  to  one-half  or  one-fourth  usually 
deprives  even  an  initially  active  serum  of  the  power  to  induce  mem- 
brane-formation. Even  when  the  undiluted  serum  is  sufficiently 
potent  to  overcome  the  inhibition  of  its  proteins  so  far  as  to  cause 
membrane-formation,  the  inhibition  is  nevertheless  operative  and  finds 
expression  in  the  prevention  of  the  subsequent  cytolysis. 

The  concentration  of  protein  in  the  medium  bathing  the  eggs  which 
is  required  to  inhibit  membrane-formation .  affords  a  quantitative 
measure  of  the  potency  of  the  fertilizing  agent.  The  more  concentrated 
a  solution  of  Saponin,  for  example,  the  greater  the  amount  of  Ovo- 
mucoid which  must  be  added  to  it  to  prevent  the  formation  of  mem- 
branes. From  this  it  is  evident  that  the  "charge"  of  membrane- 
•  forming  agent  which  the  spermatozoon  carries  into  the  egg  must  be 
less  than  that  which  is  deposited  upon  sensitized  eggs  in  an  active  serum 


458    PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 


which  induces  membrane-formation  without  previous  dilution,  for  the 
concentration  of  proteins  in  normal  undiluted  serum  is  sufficient  to 
inhibit  the  membrane-formation  succeeding  fertilization  by  sperma- 
tozoa. It  is,  however,  possible  to  increase  the  "charge"  of  membrane- 
forming  agent  in  spermatozoa  by  sensitizing  them  with  strontium 
chloride  solution  and  exposing  them  to  blood-serum  previous  to  fer- 
tilization. They  thus  accumulate  the  membrane-forming  agent  from 
the  serum  and  carry  it  together  with  their  own  membrane-forming 
agent  into  the  egg.  The  following  experiment  affords  an  illustration 
of  this  fact.  Solutions  of  ovomucoid  in  sea-water  were  prepared  con- 
taining 2,  J,  J  and  f  per  cent,  of  the  protein,  respectively  and  in 
2  c.c.  samples  of  each  of  these  solutions  were  placed  two  drops  of  a 
thick  suspension  of  the  eggs  of  Strongylocentrotus  purpuratus.  The 
sperm  from  a  male  of  the  same  species  was  divided  into  three  portions. 
The  one  portion  was  untreated  save  by  washing  in  sea- water.  A  second 
portion  was  immersed  for  four  minutes  in  f  m.  strontium  chloride  and 
then  for  one  minute  in  sea-water.  The  third  portion  was  immersed 
for  four  minutes  in  f  m.  strontium  chloride  and  then  for  four  minutes 
in  an  isotonic  undiluted  blood  serum.  These  three  samples  of  sperm 
were  then  added  to  the  eggs  contained  in  the  solutions  of  ovomucoid 
in  sea-water  described  above.  The  concentrations  of  ovomucoid 
employed  inhibited  the  formation  of  fertilization-membranes  by  the 
normal  spermatozoa  and  by  those  which  had  merely  been  immersed 
in  strontium  chloride  solution,  but,  except  in  the  case  of  the  strongest 
solution,  they  were  unable  to  prevent  the  formation  of  membranes  by 
the  spermatozoa  which  had  acquired  an  additional  charge  of  cytolytic 
substance  from  blood  serum : 


Concentration  of 
ovomucoid 
solution. 

Per  cent,  membranes 
formed  by  untreated 
sperm  after: 

Per  cent,  membranes 
formed  by  sperm  washed 
in  SrClz  and  then  in 
sea-  water  after  : 

Per  cent,  membranes 
formed  by  sperm  washed 
in  SrCla  and  then  in 
serum  after: 

15  mins. 

40  mins. 

15  mins. 

40  mins. 

15  mins. 

40  mins. 

2  per  cent.    . 

1/2     .... 
1/4     .... 
1/8     .... 

0 
0 
0 
5 

0 
0 
0 

7 

0 
0 
0 

8 

0 
0 
0 

8 

0 
20 
30 

18 

0 
30 
40 
18 

It  appears,  therefore,  that  the  cytolytic  agent  in  mammalian  blood- 
serum  when  introduced  into  the  egg-cell  together  with  the  sperma- 
tozoon, brings  about  just  the  same  effects  as  the  cytolytic  agent  in  the 
spermatozoon  itself  and  the  inference  is  thus  rendered  the  more  prob- 
able that  these  two  agents  are  similar  in  character. 

THE  EFFECT  OF  MEMBRANE-FORMING  AGENTS  UPON  THE  EGG. 

The  essential  feature  of  the  process  of  Membrane-formation  is  its 
evidently  close  relationship  to  the  phenomena  of  Cytolysis,  or  lique- 
faction and  disintegration  of  the  cell.  In  the  view  of  Loeb  it  is  cytolysis 


MEMBRANE-FORMING  AGENTS  459 

which  is  confined  to  the  cortical  layer  of  the  egg.  The  formation  of  the 
membrane  is  accompanied  by  a  very  manifest  increase  in  the  volume 
of  the  egg  which  can  only  be  accounted  for  by  an  imbibition  of  water. 
The  cytolysis  which  succeeds  membrane-formation  is  accompanied 
by  a  still  greater  swelling  and  imbibition  of  water.  This  has  been 
attributed  by  Loeb  and  von  Knaffle-Lenz  to  the  partial  liquefaction  or 
destruction  of  an  Emulsion-structure  within  the  cell  or  at  its  periphery. 
We  have  seen  (Chapter  XIII)  that  protoplasm  consists  of  an 
emulsion  of  lipoids  in  a  protein  medium  and  that  this  emulsion 
must  be  particularly  concentrated  at  the  surface  of  the  protoplasm 
owing  to  the  lowering  of  interfacial  tension  which  is  thus  brought  about. 
Any  increase  in  the  diameter  of  the  lipoidal  droplets  in  this  superficial 
layer,  or  their  coalescence,  must  lead  to  a  corresponding  increase  in 
the  width  of  the  interstices  between  them,  and  hence  to  an  enhanced 
permeability  for  water  and  salts.  The  fat-dissolving  character  of  the 
majority  of  the  cytolytic  agents  is  thus  the  origin  of  the  taking  up 
of  water  by  the  egg  which  results  in  the  physical  phenomena  of  cytoly- 
sis or  cell-liquefaction.  Any  other  agent  which  will  induce  imbibition 
of  water  will,  however,  also  bring  about  cytolysis.  For  example 
distilled  water  or  Hypotonic  Solutions  bring  about  cytolysis  because 
the  excess  of  osmotic  pressure  within  the  egg  forces  water  into  the  cell 
even  through  the  normally  narrow  interstices  of  the  cortical  layer. 
Other  agents  may  induce  cytolysis  by  altering  the  solubility  of  the 
protein  component  of  the  emulsion,  and  hence  cytolysis  may  be  induced 
in  certain  cells  by  physiologically  unbalanced  salt  solutions.  Even 
membrane  formation,  as  Lillie  has  observed,  may  be  brought  about  in 
certain  echinoderm  eggs  (Arbacia)  by  exposing  them  to  pure  solutions 
of  certain  Sodium  Salts,  and  this  effect  is  inhibited  by  an  admixture 
of  Calcium  Chloride. 

The  formation  of  the  fertilization-membrane,  therefore,  which  is 
the  first  step  in  the  stimulation  of  development  which  constitutes 
fertilization,  is  essentially  a  partial  and  arrested  cytolysis.  The  impor- 
tant question  now  presents  itself,  in  what  way  does  this  partial 
cytolysis  affect  the  chemical  processes  of  the  cell? 

One  very  decided  effect  of  fertilization  by  spermatozoa  is  enhance- 
ment of  Basal  Metabolism,  indicated  by  a  greatly  increased  consump- 
tion of  oxygen.  The  following  measurements  by  Loeb  and  Wasteneys 
illustrate  this  fact: 

CONSUMPTION  OF  OXYGEN  BY  A  GIVEN  MASS  OF  ARBACIA  EGGS. 

Mg.  of  oxygen  consumed 
Time.  per  hour. 

Before  fertilization ,      .      .      .      .0.24 

First  hour  after  fertilization 0.941 

Second  hour  after  fertilization 0.80 

Third  hour  after  fertilization 0.87 

Fourth  hour  after  fertilization 0.91 

Fifth  hour  after  fertilization .      .      1.05 

1  This  value  is  too  high,  owing  to  the  presence  of  sperm  which  were  washed  away 
before  the  next  determination  was  made. 


460     PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

During  the  period  occupied  by  the  experiment  the  eggs  had  pro- 
ceeded to  the  thirty-two  cell  stage.  The  rate  of  oxidations  does  not 
reach  its  maximum  instantaneously,  but  increases  progressively.  For 
example,  Warburg  in  comparing  the  rates  of  oxidation  in  the  8-cell  and 
32-cell  stages  found  that  they  were  in  the  ratio  of  4.2  to  6.8. 

Corresponding  with  these  facts  we  find  that  deprivation  of  oxygen 
arrests  the  processes  of  development  and  prevents  nuclear  and  cell- 
division.  The  same  effect  is  brought  about  by  Cyanides,  which  also 
arrest  cellular  oxidations  and,  in  multicellular  animals,  act  primarily 
by  reducing  tissue-respiration.  It  is  possible  to  show,  however,  that 
other  processes  besides  oxidations  are  initiated  by  fertilization,  for 
when  the  fertilized  eggs  of  Strongylocentrotus  purpuratus  are  left  in 
sea- water  free  from  oxygen  for  twenty-four  hours  at  15°  C.  they  will 
not  develop  during  that  time,  but  they  will  begin  to  develop  at  once 
if  oxygen  is  admitted.  It  is  found,  however,  that  their  development  is 
no  longer  normal,  since  they  form  abnormal  blastulse  and  never  or 
rarely  reach  the  gastrula  stage  (Loeb).  If  unfertilized  eggs  are  kept  for 
twenty-four  hours  without  oxygen  they  remain  uninjured,  and  upon  the 
addition  of  sperm  they  develop  normally  and  produce  healthy  plutei. 
The  same  result  is  obtained  if  development  is  arrested  by  potassium 
cyanide. 

It  has  been  shown  by  Loeb  in  fact  that  not  only  is  development 
arrested  by  deprivation  of  oxygen,  but  it  is  also,  to  some  extent, 
reversed.  Thus  if  development  be  initiated  by  membrane-formation 
with  butyric  acid  in  Arbaeia  eggs,  on  restoring  the  eggs  to  normal  sea- 
water  they  die  within  a  few  hours  unless  they  are  treated  with  hyper- 
tonic  sea-water;  moreover,  they  are  no  longer  fertilizable  by  sperm. 
However,  if,  instead  of  transferring  the  eggs  to  normal  sea-water, 
they  are  placed  in  sea-water  containing  sodium  or  potassium  cyanide, 
or  chloral  hydrate,  then  after  some  hours  they  no  longer  die  when  they 
are  returned  to  normal  sea-water  and,  in  fact,  may  now  be  fertilized 
by  sperm. 

The  fact  that  Chloral  Hydrate  and  other  narcotics,  as  well  as  cyanides, 
will  arrest  the  development  of  fertilized  eggs  is  a  striking  proof,  in 
itself,  that  other  chemical  phenomena  besides  oxidation  underlie 
development,  for  the  narcotics,  although  they  suppress  or  retard  the 
processes  of  cell  division  and  development  do  not  perceptibly  diminish 
the  rate  of  oxidation  in  the  egg.  The  acceleration  of  basal  metabolism 
which  occurs  in  fertilization,  therefore,  although  essential  to  develop- 
ment, is  not  the  only  essential  chemical  transformation  which  underlies 
the  process  of  development.  The  vast  majority  of  the  reactions  which 
occur  in  living  tissues  are  oxidations,  reductions,  or  hydrolyses1  and 
we  may  therefore  consider  it  probable  that  Hydrolysis  also  occurs  and 
performs  an  essential  function  in  early  development. 

It  remains  now  to  discuss  the  relative  parts  played  by  the  two 
factors  of  fertilization,  the  one  consisting  in  the  partial  cytolysis  of  the 

1  Decarboxylization  should  perhaps  be  added  to  this  list.  Deaminization  may  with 
propriety  be  classed  among  the  hydrolyses. 


MEMBRANE-FORMING  AGENTS  461 

egg  and  the  other,  which  is  also  brought  about  by  the  spermatozoon 
and  may  be  imitated  by  treating  the  eggs  with  hypertonic  sea-water. 
In  the  first  place,  as  regards  the  cytolytic  effect,  or  Membrane-formation 
it  has  been  found  that  the  characteristic  acceleration  of  Oxidations 
which  is  induced  by  complete  fertilization  is  also  induced  by  membrane- 
formation.  Thus  Warburg  compared  the  rate  of  oxidations  in  unfer- 
tilized eggs  and  in  eggs  which  had  been  fertilized  by  sperm,  and  he 
found  that  the  consumption  of  oxygen  in  the  eggs  which  had  been 
fertilized  was  10.5  times  the  consumption  of  oxygen  in  the  unfertilized 
eggs.  The  same  eggs  after  butyric  acid  treatment  consumed  9.0  times 
as  much  oxygen  as  the  unfertilized  eggs;  the  effect  of  membrane- 
formation  alone  upon  the  basal  metabolism  was  therefore  very  nearly 
equal  to  that  of  complete  fertilization.  These  experiments  were  re- 
peated by  Loeb  and  Wasteneys  who,  in  another  species  of  sea-urchin, 
found  the  ratio  of  oxygen-consumption  in  unfertilized  and  sperm- 
fertilized  eggs  to  be  1 : 4.55,  while  two  estimations  of  oxygen-consump- 
tion in  the  same  unfertilized  eggs  after  membrane-formation  by 
butyric  acid  gave  the  values  1 : 4.72  and  1 : 4.28,  indicating  that  the 
effect  of  membrane-formation  is  to  raise  the  rate  of  oxidations  to 
approximately  the  same  height  as  the  entrance  of  a  spermatozoon. 

We  have  seen  that  membrane-formation  is  essentially  a  partial  and 
arrested  Cytolysis.  That  this  is  really  the  essential  feature  in  the  proc- 
ess and  not  merely  an  incidental  phenomenon  is  shown  by  the  fact 
that  if  cytolysis  be  pushed  even  further,  by  whatever  agent  it  may  be 
caused,  the  effect  is  to  increase  the  consumption  of  oxygen  by  the  egg 
and  approximately  in  proportion  to  the  degree  of  cytolysis  which  is 
induced.  Cojnplete  cytolysis  of  the  egg  of  the  sea-urchin  can  be  caused 
by  the  addition  of  Saponin  to  the  sea-water.  Loeb  and  Wasteneys 
measured  the  rate  of  oxidations  in  a  batch  of  unfertilized  eggs  in  sea- 
water  and  they  found. that  they  consumed  0.15  mg.  of  oxygen  per  hour 
at  15°  C.  The  eggs  were  then  cytolyzed  with  saponin  and  the  amount 
of  oxygen  consumed  per  hour  at  15°  C.  determined  again.  It  was 
found  to  be  1.07  mg.  The  complete  cytolysis  of  the  eggs,  therefore, 
increased  the  rate  of  oxidation  700  per  cent.,  or  rather  more  than 
fertilization  itself.  Cytolysis  by  hypotonic  sea-water  also  causes  an 
increase  in  oxidations. 

The  second  factor  in  artificial  fertilization,  the  treatment  with  Hyper- 
tonic  Sea-water,  also  increases  the  consumption  of  oxygen  by  the  egg, 
but  only  to  a  relatively  slight  degree,  and  not  at  all  if  it  succeeds  mem- 
brane-formation whether  induced  by  a  spermatozoon  or  by  butyric 
acid.  Thus  Loeb  and  Wasteneys  obtained  the  following  results  with 
the  unfertilized  and  otherwise  untreated  eggs  of  Strongylocentrotus 
purpuratus: 

Oxygen-consumption  in 

ninety  minutes, 
Solution.  mgm. 

Normal  sea-water 0.30 

Hypertonic  sea-water  (  =  50  c.c.  sea-water  +  9  c.c.  2|  m.  NaCl 

+  KC1  +  CaCl2) 0.67 

Normal  sea-water  half  an  hour  later 0.51 

Normal  sea- water  twenty-one  hours  later 0.48 


462    PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

The  increase  in  oxygen-consumption  is  obviously  much  less  than 
that  caused  by  membrane-formation  and  it  is,  moreover,  transitory, 
falling  off  with  time  after  the  exposure  instead  of  increasing,  as  it  does 
when  the  eggs  are  normally  fertilized  or  treated  with  butyric  acid. 
On  the  other  hand  in  eggs  in  which  membrane-formation  has  been 
induced  by  butyric  acid  or  by  the  entry  of  a  spermatozoon,  no  increase 
whatever  and  no  important  decrease  in  the  rate  of  oxidations  could 
be  observed  on  treatment  with  hypertonic  sea-water.  The  corrective 
effect  of  hypertonic  solutions  in  preventing  the  death  and  disintegra- 
tion of  the  eggs  which  succeeds  membrane-formation  by  cytolytic 
agents  is  therefore  not  to  be  sought  in  an  effect  upon  oxidations.  It 
may  possibly  reside  in  an  effect  upon  underlying  hydrolyses,  for  these, 
as  we  have  seen,  will  bring  about  the  destruction  of  the  egg  if  they  are 
permitted  to  go  forward  while  the  oxidations  are  retarded  or  prevented 
by  lack  of  oxygen  or  by  cyanides,  and  hence  if  they  were  dispropor- 
tionately rapid,  even  in  eggs  in  which  oxidation  were  proceeding  they 
might  be  presumed  to  exert  a  like  deleterious  effect.  However  this 
may  be,  the  action  of  the  hypertonic  solutions  upon  the  egg  is  not 
reversible  upon  restoration  to  normal  sea-water.  The  effect  is  to  induce 
a  permanent  alteration  of  the  egg  which  renders  it  able  to  withstand 
partial  cytolysis  (membrane-formation)  without  injury.  Thus  Loeb  has 
shown  that  if  the  unfertilized  eggs  of  Strongylocentrotus  purpuratus  be 
placed  for  from  two  to  two  and  a  half  hours  in  hypertonic  sea-water 
(50  c.c.  sea-water  +  8  c.c.  2|  m.  Ringer  solution)  they  may  be  returned 
to  normal  sea-water  and  subsequent  treatment  with  butyric  acid,  even 
forty-eight  hours  later,  will  induce,  not  merely  membrane-formation, 
but  full  and  normal  development  of  the  embryo. 

THE  RELATIONSHIP  OF  PHOSPHOLIPINS  TO  THE  SYNTHESIS  OF 

NUCLEAR  MATERIAL  AND  THE  EFFECTS  OF  LECITHIN 

UPON  EARLY  DEVELOPMENT. 

The  leading  results  of  the  early  development  of  the  embryo  are, 
in  the  first  place,  the  very  great  increase  of  cellular  surface  due  to 
repeated  subdivisions  of  the  original  egg-cell  and  in  the  second  place 
an  increase  in  the  proportion  of  nuclear  to  cytoplasmic  constituents. 
The  earlier  estimations  of  Boveri  led  him  to  the  conclusion  that  the 
mass  of  nuclear  material  in  the  cells  is  doubled  at  each  cell-division, 
but  the  more  recent  estimations  of  Conklin  have  tended  to  greatly 
reduce  this  estimate,  the  average  nuclear  growth  during  cleavage 
amounting,  it  appears,  to  not  more  than  from  five  to  nine  per  cent,  for 
each  cleavage  that  occurs.  Nevertheless  there  is  a  definite  increase 
in  nuclear  material  during  the  formation  of  the  multitude  of  new  cells 
which  comprises  the  Blastula-stage  of  the  sea-urchin  and  since  during 
this  period  of  development  no  growth  of  cytoplasm  occurs,  the  cyto- 
plasm of  the  new  cells  occupying  collectively  the  same  space  as  the 
original  egg-cell,  it  is  evident  that  a  disproportion  of  nuclear  to  cyto- 


RELATIONSHIP  OF  PHOSPHOLIPINS  TO  SYNTHESIS       463 

plasmic  material  must  be  established,  a  disproportion  which  subsequent 
development  corrects. 

The  Synthesis  of  Nuclear  Material  is  a  self-accelerated  or  autocatalyzed 
phenomenon.  This  follows  from  the  fact  that  each  successive  cell 
division  occupies  about  the  same  length  of  time  as  the  preceding  one, 
but  the  number  of  nuclei  which  results  from  the  divisions  is  at  each 
division  twice  as  great  as  in  the  preceding  one.  The  rate  of  production 
of  nuclei  therefore  forms  a  geometrical  progression  in  time  intervals 
which  constitute  an  arithmetical  progression.  The  synthesis  of  nuclear 
material  thus  evidently  accelerates  the  formation  of  fresh  nuclear 
material-  (Loeb.) 

The  question  now  presents  itself  as  to  the  origin  of  the  materials 
from  which  the  nuclei  are  synthesized.  The  most  characteristic 
constituent  of  the  nucleus  is  Nucleic  Acid  which  is  built  up  by  the 
combination  of  purine  bases,  a  carbohydrate  radical  and  phosphoric 
acid.  The  derivation  of  the  first  two  of  these  components  from 
proteins  and  from  carbohydrates  previously  present  in  the  egg  is 
readily  conceivable  but  the  question  of  the  origin  of  the  phosphoric 
acid  component  suggests  several  interesting  possibilities.  In  the  first 
place  it  is  evidently  not  derived  from  the  external  medium  which 
bathes  the  cells,  for  perfectly  normal  development  will  occur  in  Van 
t'Hoff 's  Solution,  which  contains  no  phosphates.  The  phosphoric  acid 
which  is  required  for  the  synthesis  of  nucleins  must  therefore  be  derived 
from  some  constituent  of  the  egg-cell.  Two  groups  of  constituents 
present  themselves  as  abundant  sources  of  phosphoric  acid,  namely 
inorganic  phosphate  and  the  Phospholipins,  of  which  egg-lecithin  may 
be  taken  as  a  type.  Now  we  have  no  evidence  whatever  that  nucleic 
acid  can  be  synthesized  directly  from  inorganic  phosphates,  but  we 
have,  on  the  contrary,  a  great  deal  of  evidence  which  goes  to  show  that 
phospholipins  contribute  in  the  synthesis  of  nuclear  materials.  Thus, 
Miescher  has  shown  that  during  spermatogenesis  in  the  salmon  the 
"  lecithin  "-content  of  the  tissues  diminishes,  Hoppe-Seyler  has  pointed 
out  that  the  Lecithin-content  of  embryonic  tissues  is  exceptionally 
high  and  Mesernitzky  and  Plimmer  and  Scott  and  others  have  shown 
that  the  lecithin-content  of  hen's  eggs  which  is  initially  very  high, 
progressively  diminishes  during  the  development  of  the  embryo.  That 
the  same  process  occurs  in  the  development  of  the  sea-urchin  egg 
has  been  shown  by  Robertson  and  Wasteneys,  who  estimated  the  pro- 
portion of  alcohol-soluble  phosphorus  in  eggs  which  had  just  been 
fertilized  and  again  in  eggs  which  had  developed  to  blastulse  and  plutei. 
The  following  were  the  results  obtained  with  the  developing  eggs  of 
Strongylocentrotus  purpuratus: 

Percentage  of  the  total  phosphorus 
present  in  alcohol-soluble  forms. 

Stage  of  development.  Experiment  I.  Experiment  lI7 

Fertilized  eggs    .      .• .      .      39.5  46.5 

Blastulse 36.5  38.8 

Plutei 35.2  35.1 


464    PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

In  the  first  experiment  the  alcohol-soluble  phosphorus  (phospho- 
lipins)  decreased  by  one-eighth,  in  the  second  by  one-fourth,  and  this 
decrease  was  progressive.  The  experimental  evidence  from  a  diversity 
of  forms  therefore  tends  to  establish  a  relationship  between  the  disap- 
pearance of  lecithin  or  other  phospholipins  and  the  synthesis  of  nuclear 
materials. 

This  being  the  case,  great  importance  attaches  to  the  fact  that 
lecithin,  when  added  to  the  medium  in  which  sea-urchin  embryos  are 
developing,  strongly  retards  their  development.  The  fertilization- 
membrane  is  dissolved  by  lecithin,1  and  hence  if  lecithin  in  sufficient 
concentration  (0.15  per  cent.)  be  added  to  sea- water  containing  recently 
fertilized  eggs,  the  membranes  are  disintegrated  and  the  cleavage-cells 
which  have  been  formed  fall  apart,  so  that  for  merely  mechanical 
reasons  further  development  is  an  impossibility.  If  more  dilute  leci- 
thin solutions  are  employed  this  does  not  occur,  but,  at  the  same  time, 
no  effect  upon  the  rate  of  development  is  observed.  Very  different 
results  follow  the  exposure  of  the  developing  eggs  to  lecithin  solutions, 
however,  after  the  fertilization-membrane  has  in  the  normal  course  of 
development  undergone  rupture  and  liberated  free-swimming  blastulse. 
The  following  experiment  is  illustrative  of  the  phenomena  which  are 
then  observed:  The  eggs  of  a  Strong ylocentrotus  purpuratus  female 
were  divided  into  two  portions.  Both  portions  were  placed  in  sea- 
water  and  fertilized  with  sperm.  After  twenty-four  hours  both  lots 
of  eggs  had  developed  into  free-swimming  blastulse.  One  portion  was 
now  transferred  to  a  mixture  of  fifty  c.c.  of  sea- water  and  5  c.c.  of  a 
1.7  per  cent,  emulsion  of  egg-lecithin  in  ™  sodium  chloride  solution 
for  a  period  of  twenty-four  hours  and  then  returned  to  normal  sea- water. 
The  other  portion  was  left  in  normal  sea-water.  The  following  table 
shows  the  relative  development  of  the  two  portions : 

Time  after  fertilization. 

Days.  Portion  1  (controls).  Portion  2. 

1 Blastulse  Blastulse  (these  were  now  trans- 

ferred to  the  lecithin  mixture 
for  twenty-four  hours). 

2 Gastrulae  Blastulse  (these  were  now  trans- 

ferred to  normal  sea- water). 

3  .      .      .      .      .      .      Gastrulae  Blastulse. 

4  .      .      .    f .      .      .      Gastrulse  and  early       Blastulse. 

plutei 

5  •     ••'.'.     .      .     Fully  developed  Blastulse  and  25  per  cent,  gas- 

plutei  trulse. 

6 Advanced  plutei  Early    gastrulse    with     narrow 

unbranched  intestine  and 
large  clear  body-cavity. 

7  •      •      .      .      .      .     Advanced  plutei  Unchanged. 

8  •      ...      .      .      .     Advanced  plutei  The  gastrulse  are  now  retrograd- 

ing; the  intestine  has  almost 
disappeared. 
'     •      •      »      •      •      •     Unchanged  Unchanged. 

1  We  may  infer  from  this  that  the  periphery  of  the  fertilization-membrane  contains 
hpoidal  constituents  which  are  essential  to  the  integrity  of  its  structure. 


RELATIONSHIP  OF  PHOSPHOLIPINS  TO  SYNTHESIS       465 

It  is  evident  that  the  immersion  of  the  blastulae  for  twenty-four  hours 
in  a  0.15  per  cent,  solution  of  lecithin  enormously  retards  their  develop- 
ment. Especially  remarkable  is  the  fact  that  after  development  has 
actually  proceeded  to  the  gastrula  stage  it  shows  a  tendency  to  undergo 
reversion,  retracing  the  course  of  development  to  the  blastula  stage. 

If  purpuratus  eggs  are  fertilized  by  sperm  in  more  dilute  solutions  of 
lecithin  in  sea- water  (0.003  per  cent,  to  0.015  per  cent.)  the  fertilization- 
membranes  are  not  dissolved  sufficiently  rapidly  to  affect  development. 
In  these  solutions,  as  has  been  stated,  development  is  not  appreciably 
retarded  until  the  blastula  stage  is  reached,  probably  for  the  reason 
that  colloids  cannot  traverse  the  fertilization  membrane,  and  hence 
the  lecithin  cannot  penetrate  the  cells  of  the  embryo  until  the  fertiliza- 
tion membrane  has  been  ruptured.  Thereafter  development  is  very 
markedly  retarded  and  the  retardation  is  greater  the  greater  the  con- 
centration of  the  lecithin.  The  eggs  are  not  injured  by  the  lecithin, 
however,  as  they  will  ultimately  develop  to  normal  plutei  if  left  in 
these  solutions  for  a  sufficient  length  of  time. 

The  action  of  Cholesterol  is  so  very  generally  antagonistic  to  that  of 
lecithin  that  one  might  anticipate  tjiat  it  would,  as  in  fact  it  does, 
antagonize  the  effects  of  lecithin  upon  the  development  of  sea-urchin 
eggs.  If  cholesterol,  suspended  in  a  mixture  of  T^  sodium  oleate 
and  ™  sodium  chloride  be  mixed  with  lecithin  in  equal  proportions 
the  retarding  action  of  the  lecithin  upon  the  development  of  sea- 
urchin  eggs  is  almost  completely  neutralized.  The  slight  retardation 
which  is  observed  in  these  mixtures  may  be  due  to  the  Sodium  Oleate 
which  is  employed  to  keep  the  cholesterol  in  suspension,  since  sodium 
oleate  is  very  toxic  for  sea-urchin  eggs  and  embryos. 

Cholesterol  itself,  when  added  to  sea-water,  has  no  influence  upon 
the  rate  of  development  of  the  eggs.  The  emulsions  of  cholesterol 
are,  however,  coagulated  by  the  salts  in  sea-water  and  the  cholesterol 
is  completely  thrown  out  of  suspension  in  the  form  of  coarse  flocculi. 

Since  the  preparations  of  "lecithin"  employed  in  these  experiments 
simply  consisted  of  the  mixture  of  phospholipins  which  is  thrown  out 
of  an  ether  extract  of  egg-yolks  by  the  addition  of  acetone,  it  cannot 
be  definitely  decided  whether  the  effects  observed  were  in  reality  due 
to  lecithin  or  possibly  to  some  other  Phospholipin  present  as  an  admix- 
ture in  these  preparations.  The  significant  feature  of  these  results 
lies,  however,  in  the  fact  that  if  the  phospholipins  within  the  egg  itself 
and  in  other  developing  tissues  behave  similarly  to  the  "lecithin" 
from  yolks  of  eggs,  then  their  progressive  disappearance  during  nuclear 
synthesis  must  result  in  a  proportionate  diminution  of  their  retarding 
effect,  so  that  the  auto-acceleration  of  nuclear  synthesis,  alluded  to 
above,  may  wholly  or  in  part  be  due  to  the  consumption  of  phospho- 
lipins which  is  incidental  to  the  process;  progressive  removal  of  a 
retarder  being,  of  course,  equivalent  in  its  effect  to  the  progressive 
addition  of  a  catalyzer. 

30 


466     PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

THE  CHEMICAL  MECHANICS  OF  CELL-DIVISION. 

The  essential  mechanical  resultant  of  cell-division  is  the  increase  of  the 
Protoplasmic  Surfaces  which  is  brought  about.  During  the  successive 
divisions  which  an  egg-cell  undergoes  in  developing  to  the  blastula- 
stage  the  total  area  of  the  protoplasmic  surfaces  is  enormously  increased 
even  in  attaining  the  thirty-two  cell  stage,  for  example,  the  total  proto- 
plasmic surface  is  increased  by  about  three  hundred  per  cent.  Such 
an  increase  in  a  fluid  surface  necessarily  implies  either  the  performance 
of  work  by  external  forces  or  else  a  considerable  reduction  of  Superficial 
Tension. 

In  1876  it  was  suggested  by  Butschli  that  cell-division  is  brought 
about  through  an  increase  of  surface-tension,  subsequent  to  nuclear 
division,  in  the  equatorial  region  of  the  egg.  He  pointed  out  that 
substances  diffusing  from  the  nuclei  or  centrosomes  must  necessarily 
reach  their  highest  concentration  in  the  equatorial  plane  and  hence 
assuming  that  these  substances  increase  the  surface-tension  at  the 
periphery  of  the  egg,  the  most  marked  increase  would  occur  in  the 
equatorial  region  and,  as  a  consequence,  the  surface-tension  at  the  poles 
of  the  cell  would  be  less  than  that  at  the  equator. 

Such  a  conception  of  cell-division  is,  however,  manifestly  erroneous 
for  an  increase  of  interfacial  tension  at  the  equator,  such  as  Butschli 
imagines  to  occur,  implies  an  increase  in  the  molecular  attractive  forces 
at  the  equator,  and  the  fluids  of  the  cell  would  not  stream  away  from 
the  region  of  high  attraction  but  would,  on  the  contrary,  stream  toward 
it.  The  result  would  be  that  the  equatorial  surface  would  tend  to 
become  highly  curved,  as  areas  of  high  tension  in  a  fluid  always  do, 
and  the  surfaces  at  the  poles  would  become  relatively  flattened.  The 
result  would  be  the  formation  of  a  flattened  disc  with  a  highly  curved 
edge,  the  latter  representing  what  was  formerly  the  equatorial  surface 
of  the  egg.  Such  a  process  obviously  could  not  lead  to  cell-division. 
In  fact,  since  the  surfaces  which  Butschli  imagines  streaming  from  the 
nuclei  or  centrosomes  are  supposed  by  him  to  raise  the  surface-tension 
of  the  egg,  their  total  effect  could  only  be  to  diminish  the  surface  of 
the  egg  relatively  to  its  volume,  if  that  were  possible,  and  not  to  increase 
it,  which  is  what  the  forces  leading  to  cell-division  actually  accomplish. 
In  fact  no  model'  can  be  imagined  in  a  fluid  which  will  accomplish 
increase  of  surface  by  increase  of  superficial  tension.  Such  a  model 
can  be  devised  in  a  non-fluid  system,  as,  for  example  a  rubber  balloon 
subjected  to  compression  by  a  rubber  band  around  its  equatorial 
circumference.  The  equator  of  the  balloon  would  by  this  means  be 
constricted  and  the  single  sphere  would  tend  to  divide  into  two,  owing 
to  the  application  of  additional  tension  at  its  equator.  This  model  is, 
however,  in  no  way  comparable  to  a  fluid  drop,  for  it  is  characteristic 
of  the  superficial  tension  of  liquids  that  it  is  not  altered  by  diminution 
or  expansion  of  the  surface,  because  it  is  really  due  to  the  unbalanced 
attraction  of  the  underlying  molecules  of  the  liquid  for  each  other.  If 
a  cleft  is  formed  in  a  liquid  drop  the  opposite  walls  of  the  cleft  attract 


CHEMICAL  MECHANICS  OF  CELL-DIVISION  467 

one  another  and  tend  to  close  up  the  cleft  again.  If  they  fail  to  do  so 
it  can  only  be  because  the  molecular  attractions  have  been  weakened, 
i.  e.,  the  surface-tensionTiiminished.  In  a  rubber  balloon  there  is  no 
such  attraction  across  the  cleft;  the  tension  is  purely  transverse  and  is 
not  exerted  perpendicularly  to  the  surface  as  it  is  in  a  fluid,  and  so 
there  is  nothing  to  prevent  a  cleft  from  extending  deeper  and  deeper 
into  the  equator  of  the  rubber  balloon  provided  the  tension  of  the 
encircling  band  is  reduced  thereby  to  a  greater  extent  than  the  tension 
of  the  balloon  is  increased. 

In  1895  it  was  suggested  by  Loeb  that  phenomena  of  Protoplasmic 
Streaming  are  what  really  lead  to  cell-division.  He  pointed  out  that 
in  cell-division  the  protoplasm  streams  from  the  equator  of  the  cell 
in  opposite  directions  toward  the  two  nuclei;  the  violence  of  these 
streaming  movements,  he  suggested,  brings  about  the  mechanical 
separation  of  the  two  cells. 

The  streaming  of  protoplasm  from  the  equator  toward  the  poles 
suggests  that  the  phenomenon  antecedent  to  cell-division  is  a  diminu- 
tion of  surface-tension  in  the  equatorial  region  and  not,  as  Biitschli 
suggests  an  increase.  That  such  equatorial  diminution  in  surface- 
tension  will  bring  about  the  division  of  droplets  into  two  is  very  readily 
shown  by  means  of  the  following  simple  experiment : 

The  formation  of  Soaps  at  the  surface  of  oil-droplets,  results  as  we 
have  seen  in  Chapter  XIII,  in  a  diminution  of  the  surface-tension  of  the 
droplets;  if  the  formation  of  soap  is  local,  that  part  of  the  surface  upon 
which  the  soap  is  formed  tends  to  spread.  Since  commercial  olive  oil 
almost  invariably  contains  traces  of  Fatty  Acid  the  result  of  bringing  an 
alkali  in  contact  with  a  drop  of  such  oil  will  be  the  formation  of  soap  at 
the  points  of  contact.  If,  now,  a  drop  of  Olive  Oil  which  is  not  too 
large  (about  2  to  3  mm.  in  diameter)  be  floated  on  a  layer  of  water,  and 
a  thread  saturated  with  tenth  normal  alkali  (NaOH  or  K0H)  be  brought 
gently  into  contact  with  a  diameter  of  the  drop,  the  almost  immediate 
effect  is  the  division  of  the  drop  into  two.  The  phenomena  accompany- 
ing this  division  are  perfectly  characteristic.  Instantly  the  edges  of 
the  drop  (the  ends  of  the  diameter  along  which  the  thread  lies)  recede 
from  the  thread,  forming  a  notch  at  each  end  of  the  diameter,  and 
violent  streaming-motions  occur  at  the  surface  away  from  the  thread 
and  toward  the  opposite  poles  of  the  drop.  These  streaming  move- 
ments may  be  so  violent  as  to  rotate  the  droplets  into  which  the  drop 
divides  through  as  much  as  360°.  If  the  division  does  not  occur  too 
rapidly  the  streaming  may  result  in  the  two  droplets  being  connected 
by  a  thread  of  oil,  which  may  be  central  or  to  one  side,  and  it  may  then 
be  clearly  seen  that  the  mechanism  which  brings  about  the  snapping  of 
this  thread  is  the  violent  streaming  in  opposite  directions  which  takes 
place  in  the  drops.  Phenomena  almost  exactly  resembling  those 
described  by  Loeb  in  dividing  ova  may  readily  be  observed  (Fig.  30). 
Frequently,  also,  processes  resembling  Pseudopodia  are  thrown  out  by 
the  droplets  in  the  act  of  their  division. 

The  segmentation  of  the  drop  is  not  due  to  mechanical  division  by 


408     PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

the  thread,  for,  in  the  first  place,  the  streaming  phenomena,  etc.,  are 
obviously  attributable  to  soap-formation,  and,  in  the  second  place,  the 
phenomena  observed  when  a  thread,  un wetted  save  with  water,  is  laid 
across  the  drop  are  quite  different  from  those  described  above.  The 
drop  of  oil  adheres  to  the  thread  and  forms  an  elongated  ellipsoid,  its 
long  axis  coinciding  with  the  thread;  in  fact  the  drop  of  oil  assumes 
somewhat  the  form  which' the  cell  would  assume  were  the  phenomenon 
subsequent  to  nuclear  division,  as  Biitschli  imagines,  an  increase  in 
surface-tension  at  the  circumference  of  the  equatorial  layer. 

Similar  phenomena  may  be  obtained  with  submerged  droplets, 
formed  by  adding  chloroform  to  the  oil  to  increase  its  specific  gravity, 
or  by  droplets  immersed  in  a  column  of  salt  solution  of  varying  con- 
centration the  lower  layers  being  saturated,  so  that  the  drop  floats 
midway  without  sinking  or  rising,  only  in  this  case  stronger  alkali 
must  be  used  because  the  greater  part  of  it  is  washed  off  the  thread  in 
passing  it  down  to  the  drop  through  the  upper  layers  of  water  or  salt 
solution. 


12  3  4 

Fie.  30. — Drawings  of  a  case  of  cell-division  in  artificial  parthenogenesis  (sea-urchin 
egg)  illustrating  the  underlying  phenomenon  of  streaming.  "The  division  began  on 
one  side  (1)  and  the  protoplasm  then  flowed  in  the  direction  of  the  arrows  (2)  in  oppo- 
site directions  toward  the  two  nuclei.  The  connecting-piece  becomes  empty  of  proto- 
plasm and  only  the  pigmented  solid  surface  film  is  left  (3)  and  finally  this  also  disappears 
(4)."  (After  Loeb.) 

The  action  of  alkalies  is  not  confined  to  those  mentioned  above  but, 
apparently,  the  division  and  accompanying  phenomena  can  be  brought 
about  by  means  of  threads  dipped  in  all  bases  which  form  soaps  with 
fatty  acids.  Thus  tenth-normal  potassium  hydroxide  or  sodium  hydrox- 
ide and  a  saturated  solution  of  calcium  hydroxide,  all  bring  about  the 
division,  although  the  division  when  calcium  hydroxide  is  used  is  less 
rapid  than  when  tenth-normal  sodium  or  potassium  hydroxide  are 
employed,  because  the  concentration  of  a  saturated  solution  of  calcium 
hydroxide  is  only  about  twentieth  normal.  The  division  and  accom- 
panying phenomena  are  also  elicited  in  a  marked  degree  by  threads 
dipped  in  Choline. 

Not  only  the  bases,  but  the  soaps  themselves  bring  about  the 
division;  thus  if  a  thread  smeared  with  Choline  Oleate  be  laid  across  the 
diameter  of  a  drop  of  olive  oil,  the  division  of  the  drop  will  occur, 
although  more  slowly  than  when  choline  itself  is  used.  This  shows  that 
the  action  of  these  bases  is  due  to  the  soap  which  is  formed  when  they 
come  into  contact  with  the  oil  and  not  to  hydroxyl  ions. 

Now  we  have  seen  that  the  phosphoric  acid  component  of  the 
nucleic  acid  molecule  is  probably  derived,  during  nuclear  synthesis, 
from  Lecithin  or  similar  Phospholipins.  The  decomposition  of  lecithin 
for  this  purpose  must  lead  to  the  setting  free  either  of  Choline  itself 


FORMATION  OF  MONSTROSITIES  469 

or  of  a  soap  of  choline  or  some  other  nitrogenous  base,  formed  by  com- 
bination with  the  fatty-acid  radicals  of  the  phospholipin.  Immediately 
following  the  division  of  the  cell-nucleus  into  two,  which  precedes  by 
a  definite  interval  the  division  of  the  cell,  we  may  suppose  an  active 
synthesis  of  nuclear  materials  to  be  occurring  in  the  two  nuclear  regions. 
Hence,  in  these  localities,  provided  that  the  above  hypothesis  be  correct 
choline  or  some  other  nitrogenous  base  would  be  set  free.  If  now,  choline 
be  liberated  at  both  nuclei  and  diffuses  from  each  nucleus  equally  in 
all  directions  its  maximal  concentration  must  obviously  occur  in  the 
equatorial  plane  at  right  angles  to  the  line  joining  the  two  nuclei. 
We  have  seen  that  choline,  when  applied  to  the  diameter  of  a  droplet  of 
liquid  immiscible  with  water  (provided  soap  is  formed)  results  in  the 
division  of 'the  drop  along  that  diameter.  It  is  possible  that  choline, 
set  free  in  nuclein  synthesis,  brings  about,  in  a  similar  manner,  the 
division  of  the  cell,  through  the  formation  of  soaps  in  the  equatorial 
plane,  either  through  combination  with  fatty  acids  in  the  cytoplasm,  or 
else  through  its  having  been  liberated  in  the  neighborhood  of  the  nuclei 
in  combination  with  one  or  more  of  the  oleic,  stearic  or  palmitic  acid 
groups  of  the  lecithin  molecule. 

It  is  not  even  necessary  to  presuppose  an  actual  separation  of  the 
two  nuclei;  it  is  only  necessary  to  suppose  that  the  nuclein  synthesis 
occurs  with  greater  rapidity  at  opposite  poles  than  elsewhere  within 
the  nucleus  in  order  to  understand  how  nuclear  division  may  be  brought 
about  by  essentially  the  same  mechanism  as  that  which  brings  about 
cell-division  itself. 


ARTIFICIAL  TWIN-FORMATION  AND  THE  FORMATION  OF 
MONSTROSITIES. 

In  the  normal  development  of  the  egg  the  early  cleavage-cells, 
although  distinct  and  separated  from  one  another  by  a  definite  inter- 
face, nevertheless  remain  in  close  apposition  to  one  another,  So  long 
as  this  is  the  case  a  single  embryo  develops.  If,  however,  the  first  two 
cleavage-cells  chance  to  fall  apart  and  cease  to  remain  in  their  normal 
closeness  of  apposition  then  each  of  the  cells  develops  into  a  separate 
and  complete  embryo  and  twins  are  formed  from  a  single  egg;  these  are 
probably  similar  in  origin  to  the  "identical  twins"  which  are  occasion- 
ally encountered  among  higher  animals  and  man. 

It  has  been  found  by  Loeb  that  the  separation  of  the  first  cleavage- 
cells  may  be  brought  about  in  over  ninety  per  cent,  of  fertilized  sea- 
urchin  eggs,  provided  they  are  merely  exposed,  for  some  time  after 
the  first  cell-division,  to  an  artificial  sea-water  differing  from  normal 
sea-water  in  the  lack  of  any  one  of  the  constituents  Sodium,  Potassium 
or  Calcium.  This  change  in  the  composition  of  the  surrounding  saline 
medium  apparently  so  alters  the  consistency  of  the  surfaces  of  the 
cleavage-cells  that  they  no  longer  adhere  to  one  another.  It  may  be 
noted  that  as  the  fertilization  membrane  still  surrounds  both  of  the 
cleavage-cells  and  the  composition  of  the  external  saline  mixture  can 
nevertheless  affect  the  surfaces  of  the  eggs,  the  fertilization  membrane 


470     PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

must  be  freely  permeable  for  inorganic  salts,  although,  as  we  have 
seen,  it  is  not  permeable  for  colloids. 

The  two  embryos  develop  side  by  side  within  the  fertilization- 
membrane  and  form  swimming  blastulse.  At  the  usual  time  the 
membrane  bursts  and  sets  the  free-swimming  embryos  at  liberty. 
They  are  smaller  than  normal  embryos  of  the  same  age  but  otherwise 
differ  in  no  respect  from  embryos  which  arise  in  the  usual  way. 

The  opposite  phenomenon,  that  of  fusion  of  two  egg-cells  may  also 
be  brought  about  in  a  certain  percentage  of  cases  by  treatment  of  the 
eggs  with  alkaline  sea- water.  This  results  in  the  production  of  gigantic 
embryos.  Even  at  later  stages  of  development  similar  fusions  may  be 
made  to  occur.  Thus  Stockard  has  found  that  fusion  of  the  cells 
which  subsequently  give  rise  to  the  eyes  of  a  fish  embryo,  Fundulus 
heteroclitus,  may  be  caused  by  immersing  the  embryos  at  a  certain  stage 
of  their  development  in  sea-water  containing  an  excess  of  Magnesium. 
The  effect  of  this  is  to  cause  the  development  of  fishes  provided  only 
with  a  single  cyclopean  eye.  The  origin  of  these  ajid  other  like  phe- 
nomena is  to  be  sought  in  the  influence  which  the  composition  of  the 
surrounding  medium  exerts  upon  the  consistency  of  the  protein  and 
lipoid  emulsions  within  and  at  the  surfaces  of  the  cells. 

REFERENCES. 
GENERAL: 

Harvey:     Science  N.  S.,  1909,  30,  p.  694.     Jour,  Exp.  Zool.,  1910,  8,  p.  355.     Biol. 

Bull.,  1909-10,  18,  p.  269  (consult  for  literature) ;  1914,  27,  p.  237. 
McClendon:     Science  N.  S.,  1910,  32,  pp.  122  and  317. 
Littie,  F.  R.:     Jour.  Exp.  Zool.,  1913,  14,  p.  515. 

Loeb:     Artificial  Parthenogenesis  and  Fertilisation,   Chicago,    1913.     The  Organ- 
ism as  a  Whole,  New  York,  1916. 
Godlewsk't:     Physiologie  der  Zeugung,  in  Winterstein's  Handbuch  der  Vergleichen- 

den  Physiologie,  Jena,  1914,  Pt.  2,  3,  p.  457. 
Lillie,  R.  S.:     Jour.  Biol.  Chem.,  1914,  17,  p.  121. 
CYTOLYTIC  AND  MEMBRANE-FORMING  AGENT  IN  BLOOD: 

Loeb,  J.:     University  of  California  Pubs,  in  Physiol.,    1907,    3,   p.   57.     Pfliiger's 

Arch,  1908,  124,  p.  37.     Arch.  f.  Entwicklungsmech.,  1910,  Pt.  2,  30,  p.  44. 
Moore:'    University  of  California  Pubs,  in  Physiol.,  1912,  4,  p.  91. 
Robertson:     Arch.  f.  Entwicklungsmechan.,  1912-13,  35,  p.   64;     1913,  37,  p.   29. 

Jour.  Biol.  Chem.,   1912    12,  p.  163. 
Clark:     Ibid.,  1918,  35,  p.  253. 
SYNTHESIS  or  NUCLEAR  MATERIAL: 

Meischer:     Histochemische  und  Physio! ogische  Arbeiten,  Leipzig,  1897. 
Loeb:     Proc.  7th  Int.  Zool.  Congress  in  Boston,  1907,  Biol.  Centr.,  1910,  30,  p.  437. 
Godlewski:    Arch.  f.  Entwicklungsmech.,   1908,  26,  p.  278. 
Plimmcr  and  Scott:     Trans.  Chem.  Soc.,  London,  1908,  93,  p.  1700. 
Conklin:     Jour.  Exp.  Zool.,  1912,  12,  p    1. 

Robertson  and  Wasteneys:     Archiv.  fur  Entwicklungsmech.,  1913,  37,  p.  485. 
Robertson:     Ibid.,  1913,  37,  p.  497. 

Browder:     Univ.  of  Calif.  Pubs.  Physiol.,  1915,  5,  p.  1. 
CHEMICAL  MECHANICS  OF  CELL-DIVISION: 

Robertson:     Arch.  f.  Entwicklungsmech.,  1909,  27,  p.  29;  1911,  32,  p.  308;  1912- 

13),  p.  692. 
McClendon:     Am.  Jour.  Physiol.,  1910,  27,  p.  240.     Arch.  f.  Entwicklungsmech.,  1, 

1912,  34,  p.  263. 
FORMATION  OF  TWINS  AND  MONSTROSITIES: 

Stockard:     Jour.  Exp.  Zool.,  1907,  4,  p.  165;  1909,  6,  p.  286.     Am.  Jour.  Anat., 

1910,  10,  p.  369. 

Loeb:     Arch.  f.  Entwicklungsmech.,  1909,  27,  p.  119.     Biol.  Bull.,  1905,  29,  p.  50. 
McClendon:     Am.  Jour.  Physiol.,  1911-12.  29.  p.  289. 


CHAPTER  XX. 

PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION: 

GROWTH. 

GENERAL  CHARACTERISTICS  OF  THE  GROWTH-PROCESS. 

Regarded  from  the  chemical  point  of  view  the  growth  of  animals 
consists,  essentially,  in  the  transformation  of  simple,  unorganised 
Foodstuffs,  such  as  water,  the  inorganic  salts,  fats,  carbohydrates, 
amino-acids,  and  so  forth  into  new  chemical  entities  which,  collectively 
regarded,  form  the  organised  protoplasm  of  the  animal  tissues. 
Growth,  therefore,  involves  the  synthesis  of  a  variety  of  chemical 
compounds  in  due  proportion  and  succession  to  one  another. 

This  process  obviously  does  not  take  place  with  uniform  velocity 
throughout  life.  It  is  not  at  all  unusual,  for  example,  for  an  infant  to 
grow,  during  the  first  months  succeeding  birth,  at  the  rate  of  two 
pounds  per  month.  Were  this  rate  of  growth  maintained,  then  at 
twenty  years  of  age  we  would  weigh  in  the  neighborhood  of  five 
hundred  pounds. 

Nevertheless  the  process  of  growth  is  not  one  which  undergoes  a 
uniform  retardation,  diminishing  in  velocity  by  a  uniform  proportion 
per  annum.  On  the  contrary,  the  growth  of  children,  and  of  animals, 
takes  place  in  spurts,  separated  more  or  less  distinctly  from  one 
another  by  periods  of  relatively  languid  growth.  Thus  the  rate  of 
growth  in  utero  during  the  first  half  of  gestation  is  so  slow  that  prior 
to  this  period  the  weight  of  the  human  foetus  is  inappreciable  in  com- 
parison with  that  of  the  mother.  This  period  of  slow  growth  is  suc- 
ceeded by  the  extraordinarily  rapid  accretion  of  tissue  which  charac- 
terises development  duiing  the  months  immediately  prior  to  and 
succeeding  delivery.  A  definite  slackening  of  growth  occurs,  however, 
toward  the  end  of  the  first  year  of  extrauterine  life,  and  this  slowing 
down  of  growth  is  not  an  artefact,  dependent  upon  weaning,  since  it 
occurs  just  as  strikingly  in  bottle-fed  infants.  This  resting  period  is 
succeeded  by  the  relatively  rapid  growth  of  the  third,  fourth,  and  fifth 
years  Another  pause  or  slackening  of  growth  succeeds  this,  to  be 
followed  by  the  energetic  growth  which  accompanies  adolescence. 

The  growth  of  man,  therefore,  consists  of  periods  of  rapid  and  slow 
growth  which  alternate  with  one  another,  and  if  we  plot  the  growth  in 
any  dimension,  for  example  the  growth  in  weight,  on  "  coordinate  paper" 
so  that  the  weights  are  measured  vertically  and  ages  horizontally,  we 
obtain  a  diagrammatic  picture  of  the  growth-process  which  is  not  a 


472  PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

straight  line,  nor  even  a  single  curvilinear  sweep,  like  the  outline  of  a 
parabola  or  of  the  logarithmic  curve  which  represents  the  progress  of 
the  ordinary  type  of  chemical  reaction.  On  the  contrary,  our  diagram 
reveals  distinct  waves  or  large  oscillations  in  the  growth-process  and 
resembles,  as  a  matter  of  fact,  the  diagram  which  may  be  obtained  by 
superimposing  three  S-shaped  curves  upon  one  another  in  such  a 
manner  that  their  adjacent  extremities  merge  into  one  another. 

These  waves  or  oscillations,  or  "Growth-cycles,"  as  we  may  term 
them,  are  not  accidental.  They  are  easily  distinguishable  from  the 
relatively  slight  irregularities  or  fluctuations  of  growth  which  every 
individual  child  or  animal  will  display  more  or  less  frequently  during 
its  development.  They  are  distinguishable  from  such  accidental 
fluctuations  because  they  occur  at  very  nearly  the  same  places  in  the 
growth-curve  of  every  normal  child,  and  in  the  average  growth-diagram 
constructed  from  the  data  supplied  by  a  large  number  of  individuals, 
these  large  oscillations  reveal  themselves  very  distinctly,  while  the 
accidental  and  individual  fluctuations  cancel  out  and  disappear  in  the 
average  diagram  because,  in  the  long  run,  if  we  take  a  sufficient  number 
and  variety  of  individuals  into  account,  just  as  many  of  these  accidental 
fluctuations  will  be  positive  (i.  e.,  supernormal  in  weight)  as  negative 
(i.  e.,  subnormal  in  weight).  But  the  large  fluctuations,  or  Growth- 
cycles,  remain  unaffected  in  magnitude  and  position,  and  only  appear 
more  definitely  in  the  diagram  the  greater  the  number  of  individuals 
which  we  measure  or  weigh. 

In  the  Growth  of  Man  there  are,  in  all,  three  distinguishable  growth- 
cycles  which  are  superimposed  upon  one  another.  Each  cycle  begins 
with  a  period  of  relatively  slow  growth,  followed  by  a  period  of  very 
rapid  growth,  and  culminating,  with  the  termination  of  the  cycle,  in  a 
period  of  slackening  growth  again.  In  the  case  of  the  first  two  cycles 
this  slackening  of  growth  is  followed  by  the  fresh  spurt  or  acceleration 
due  to  the  succeeding  cycle.  In  the  case  of  the  third  or  adolescent 
cycle  of  growth,  the  period  of  slackened  growth-velocity  insensibly 
merges  into  the  period  of  relatively  stationary  development  which  we 
recognize  as  the  adult  condition.  This  developmental  stasis  may  be 
interrupted,  however,  by  the  repair  incident  to  the  replacement  of 
tissue  which  has  been  injured  or  destroyed,  while  even  in  the  absence 
of  such  Regenerative  Growth  a  vigorous  and  abnormal  growth  may 
occur,  the  growth,  namely,  of  Malignant  Tumors,  which  we  may  possibly 
interpret  as  constituting  the  superposition  of  a  fourth,  and  physio- 
logically abnormal  cycle  of  growth  upon  the  third  and  normally  final 
cycle  in  the  development  of  man. 

Not  only  the  growth  of  man,  but  also  the  growth  of  every  mammal 
which  as  yet  has  been  carefully  investigated  appears  to  consist  of 
three  more  or  less  easily  distinguishable  cycles  of  growth.  The  growth 
of  the  Guinea-pig  at  first  appeared  to  consist  of  only  two  difficultly 
distinguishable  cycles,  but  the  investigations  of  Read  have  shown  that 
in  this  mammal  the  first  growth-cycle  is  actually  completed  in  utero, 


GENERAL  CHARACTERISTICS  OF  GROWTH-PROCESS       473 


instead  of  being  interrupted  when  half-completed  by  birth,  as  it  is  in 
human  beings.  Corresponding  to  this  we  find  that  the  guinea-pig  is 
born  at  a  much  more  advanced  stage  of  development  than  man  or  the 
rat  or  mouse;  their  eyes  are  open,  they  have  a  full  coat  of  hair,  are  able 
to  choose  and  eat  their  own  food  and  may  be  weaned  altogether  within 
a  few  days  after  delivery.  The  very  general  occurrence  of  three  growth 
cycles  in  mammalian  development  renders  very  inviting  the  supposi- 
tion that  they  are  referable  to  the  existence  of  three  Embryonic  Layers, 
from  one  or  other  of  which  all  the  tissues  of  the  adult  are  ultimately 
derived,  but  for  this  hypothesis  there  are  as  yet  lacking  the  necessary 
experimental  and  anatomical  proofs. 

In  the  accompanying  figure  (Fig.  31)  are  compared  the  growth- 
diagrams  of  human  males  of  British  birth  and  parentage  and  of  male 


60  KILOGRAMS 


WEEKS 


FIG.  31. — Growth  of  human  males.  (Con- 
structed from  the  data  obtained  by  the  British 
Anthropometric  Committee.) 


Growth  of  male  white  mice. 


white  mice.  The  resemblance  between  the  two  curves,  allowing  for 
the  difference  of  the  time-units  employed,  is  of  a  very  striking  character. 
The  only  notable  difference  lies  in  the  relatively  marked  delay  of  the 
third,  or  adolescent  growth-cycle  in  man  as  cpmpared  with  the  mouse, 
the  possible  origin  of  which  will  be  discussed  subsequently. 

These  Growth-cycles,  so  definitely  situated  in  the  curve  of  growth, 
and  so  invariable  in  their  occurrence  that  they  may  be  clearly  recog- 
nised in  the  growth  of  mice  no  less  than  in  the  growth  of  man,  must 
have  some  very  definite  physiological  significance,  and  since,  as  we  have 
seen,  growth  is  essentially  a  chemical  process  resulting  in  the  synthesis 
of  living  tissue  from  inanimate  materials,  these  growth-cycles  must 
have  a  chemical,  no  less  than  a  physiological  significance.  The  general 
similarity  of  the  fundamental  phenomena  of  growth  in  all  living  forms 


474  PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

is  strikingly  revealed  by  the  fact  that  the  curves  of  growth  obtained  in 
Plants  and  even  in  the  multiplication  of  Bacteria  are  essentially  similar 
in  character  to  those  obtained  in  animals.  As  a  rule,  however,  the 
growth  of  a  plant  or  of  a  colony  of  bacteria  displays  evidence  of  only  a 
single  growth-cycle. 

Each  of  these  growth-cycles  is  approximately  symmetrical  about  its 
center,  that  is,  on  either  side  of  the  moment  of  most  rapid  growth;  in 
other  words  the  second  half  of  the  S-curve  reproduces  in  the  reverse 
order  the  characteristics  of  the  first  half.  We  have,  then,  in  each 
growth-cycle  considered  by  itself,  a  chemical  process  which  begins 
relatively  slowly,  increases  progressively  in  velocity  until  it  is  about 
half  completed,  and  then  slows  off  to  its  termination.  The  inquiry 
now  immediately  presents  itself  whether  any  chemical  processes  of 


Body-iueight.  ^f^  Amount  transformed 


FIG.  32 — Comparison  of  the  curve  of  growth  of  the  white  rat  (constructed  from  data 
collected  by  Donaldson)  with  chemical  reaction  curves. 

this  general  character  are  known  to  occur  elsewhere  than  in  the  build- 
ing up  of  tissue  by  a  growing  plant  or  animal?  As  a  matter  of  fact, 
chemical  transformations  of  this  character  are  abundant  and  they  are 
those  in  which  one  or  more  of  the  products  catalyzes  the  further 
progress  of  the  reaction.  We  have  already  in  preceding  chapters  had 
occasion  to  dwell  upon  a  number  of  chemical  phenomena  which  occur 
in  living  tissues  and  elsewhere  which  belong  to  this  category;  it  will 
merely  be  necessary  therefore  to  refer  in  passing  to  the  analogies 
afforded  by  the  hydrolysis  of  castor-oil  in  the  seeds  of  Ricinus,  the 
hydrolysis  of  cane-sugar  by  boiling  neutral  water,  the  decomposition 
of  methyl  acetate  by  water,  the  oxidation  of  metals  and  of  a  variety  of 
organic  materials,  and  the  chemical  transformations  which  accompany 
and  underlie  the  performance  of  muscular  work.  In  all  of  these  various 


GENERAL  CHARACTERISTICS  OF  GROWTH -PROCESS       4?5 

processes  one  or  more  of  the  products  of  the  reaction  is  endowed  with 
the  property  of  facilitating  the  further  progress  of  the  reaction.  Such 
transformations  are  designated  Autocatalyzed  Reactions  (Fig.  32). 

The  fact  that  each  growth-cycle  begins  slowly  and  progressively 
increases  in  velocity  until  the  moment  of  maximal  growth-velocity  is 
attained  at  the  center  of  the  cycle  is  sufficient  in  itself  to  show  that  the 
process  of  growth  is  autocatalyzed,  whatever  the  mechanism  of  the 
self-acceleration  may  be.  The  resemblance  of  the  process  of  growth 
to  the  transformations  in  an  autocatalyzed  reaction  is  not  merely 
superficial,  however,  but  extends  even  to  quantitative  details. 

It  will  be  recollected  that  the  relationship  between  the  extent  of 
transformation  and  the  time  in  an  ordinary  Monomolecular  Chemical 
Reaction  is  expressed  by  the  equation: 

Velocity      =     k(a    —  x) 

where  "a— x"  is  the  amount  of  the  original  material  which  is  as  yet 
untransformed  and  "k"  is  a  constant,  specific  for  the  particular  reac- 
tions under  consideration.  The  effect  of  catalyzers  upon  such  a  reac- 
tion is  to  multiply  the  value  of  "k"  by  a  quantity  which  is  proportional 
to  the  amount  of  catalyzer  present.  Now  in  an  autocatalyzed  reaction 
the  amount  of  catalyzer  which  is  present  is  proportional  to  the  mass  of 
the  product  of  the  reaction,  that  is,  to  "x."  The  equation  for  an 
Autocatalyzed  Monomolecular  Reaction  becomes,  therefore: 

Velocity      =     kx(a   —  x) 

which,  when  integrated,  yields  the  equation: 

Iog10  -  =     ka(t    -  ti) 

where  t  is  the  time  from  the  beginning  of  the  measurements  and  ti  is 
the  time  at  which  the  reaction  is  half  completed,  i.  e.,  the  center  of  the 
autocatalytic  curve. 

The  applicability  of  this  equation  to  the  growth  in  numbers  of 
Bacteria  in  a  limited  quantity  of  culture  medium  has  been  established 
by  McKendrick.  It  is,  however,  not  less  applicable  to  relatively 
complex  phenomena  of  Human  Growth.  The  juvenile  and  adolescent 
cycles  of  growth  in  man  are  rather  closely  interfused,  so  that  their 
separation  into  individual  cycles  is  a  difficult  and  uncertain  matter. 
The  infantile  cycle,  however,  is  rather  definitely  separated  from  the 
remainder  of  the  human-growth  curve  by  a  rather  long  period  or  "  pla- 
teau" of  relatively  slow  growth.  The  Infantile  Growth -cycle,  therefore, 
at  any  rate  for  the  first  ten  months  succeeding  birth,  presents  the 
relatively  uncomplicated  characteristics  of  a  single  cycle  of  growth  to 
which  the  above  equations  may  be  applied.  In  the  following  compari- 
sons of  the  theoretical  values  calculated  from  the  equation 

Iog10  — - —      =     ka(t    -  ti) 


476  PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

with  the  average  values  actually  obtained  from  weighings  of  large 
numbers  of  infants  the  constants  "  a,"  "  k"  and  "  t,"  are  calculated  from 
all  of  the  observations  by  the  "  method  of  least  squares.  In  this  way, 
for  example,  we  find  that  the  growth  of  British  male  infants  born  in 
South  Australia,  during  the  first  nine  months  succeeding  delivery,  is 
expressed  by  the  formula 

Iog10-  =     0.136(t    -   1.66) 

341 .5    —  x 

time  being  reckoned  in  months  from  birth  and  weights  in  ounces  avoir- 
dupois. In  the  following  table  the  observed  weights  at  the  various 
ages  are  compared  with  those  calculated  from  this  formula: 


Age  of  infant 
in  months. 

0 
1 
2 
3 

4 
5 
6 

7 
8 


The  equation  to  the  curve  of  growth  for  the  first  nine  months  of  the 
extra-uterine  life  of  South  Australian  females  is  found  to  be: 


SOUTH  AUSTRALIAN  MALES. 

Weight  in  ounces, 
int 

3.                                                                                         Observed. 
127 

Calculated. 
127 
156 
180 
206 
230 
254 
273 
288 
301 
311 

,      .      .      .      .      .      .                                                  155 

...'*.      .      .      .     .                                   187 

206 

.•••'.      .      .      .      .      .                                    224 

.      .      ....      ...                        254 

.      .      .      .      .    '                                            270 

«     ;     ...     .     .     .     .'                      287 

.      .      .      .      .      ,      «      4                       300 

311 

and  in  the  following  table  the  observed  weights  at  various  ages  are 
compared  with  those  calculated  from  the  formula: 


SOUTH   AUSTRALIAN   FEMALES. 


Age  of  infant 
in  months. 

0      ... 

Weig 

ht  in  ounces. 

Observed. 
121 

Calculated. 
121 
142 
164 
187 
209 
230 
249 
267 
282 
295 

1      ... 

153 

2     .... 

168 

3     .... 

188 

4     .... 

209 

5     .      . 

224 

6     .      . 

253 

7     ... 

263 

8     ... 

270 

9     ... 

300 

A  similar  comparison  follows  for  British  infants  born  in  England: 
The  equation  to  the  infantile  growth-cycle  during  the  first  nine  months 
m  males  is  represented  by  the  formula: 


io 

«5  lo     — 


0.127(t    -   1.46) 


GENERAL  CHARACTERISTICS  OF  GROWTH-PROCESS       477 

BRITISH  MALES. 

Weight  in  ounces. 
Age  of  infant 


in  months.                                                                                           Observed.  Calculated. 

1 147  148 

2 169  171 

3  ........< 194  194 

4 .   .  219  216 

5 234  235 

6 252  252 

7 269  266 

8 276  277 

9 .   283  287 

The  equation  for  the  same  period  in  females  is  represented  by: 

logio =     0.106(t    -   1.54) 


BRITISH  FEMALES. 


Age  of  infant 
in  months. 

1 

2 


Weight  in 

ounces. 

i.                                                                                           Observed. 
.      .      143 

Calculated. 
146 

160 
180 

165 

184 

202 
218 

202 

218 

.      .      235 

233 

253 

247 

258 

259 

265 

269 

In  all  cases  it  will  be  seen  that  the  agreement  between  the  observed 
and  the  calculated  weights  is  extremely  close;  in  fact  such  consonance 
between  the  quantitative  demands  of  a  theoretical  equation  and  the 
experimental  estimations  is  not  frequently  obtained  even  in  experi- 
ments conducted  in  laboratory-glassware.  The  probable  reasons  for 
the  extreme  regularity  observed  lie  in  the  first  place  in  the  large  number 
of  measurements  from  which  each  average  weight  is  computed  and  in 
the  second  place  in  the  excellent  conditions  of  thermostasis  which  the 
body  of  a  warm-blooded  animal  affords. 

Even  in  such  complex  Metazoa  as  man,  therefore,  the  process  of 
growth  in  an  individual  growth-cycle  appears  to  be  determined  and 
governed  by  the  simple  law  which  is  characteristic  of  an  Autocatalyzed 
Monomolecular  Reaction.  It  will  at  once  occur  to  the  reader,  however, 
that  the  process  of  growth,  taken  as  a  whole,  cannot  possibly  be  of 
this  simplicity,  for  in  the  construction  of  the  simplest  of  the  multi- 
tudinous constituents  of  tissues  a  variety  of  parallel  and  successive 
chemical  reactions  must  as  a  rule  contribute  to  the  result.  The 
diversity  of  interdependent  chemical  phenomena  involved  in  the 
building  up  of  an  organism  so  complicated  as  ourselves  must  be  almost 
unimaginably  great.  How,  then,  can  a  reaction-formula  characteristic 
of  a  single  and  uncomplicated  transformation,  peculiar  only  in  produc- 
ing its  own  catalyzer,  apply  to  the  quantitative  outcome  of  such  a 
bewildering  tissue  of  chemical  events? 


478     PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

The  answer  to  this  question  is  undoubtedly  to  be  sought  in  the  fact 
that  in  any  system  of  interdependent  chemical  transformations  the 
slowest  reaction  in  the  series  governs  the  velocity  of  the  whole.  On  the 
hither  side  of  the  slowest  reaction  all  the  raw  materials  for  subsequent 
processes  must  accumulate  and  await  the  elaboration  of  the  products 
which  they  utilize,  while  on  the  far  side  of  the  slowest  reaction  the 
subsequent  processes  are  retarded  to  its  pace  by  the  consumption  of 
their  substrates.  The  slowest  reaction  in  any  chain  of  chemical 
processes  is  the  Master-reaction  which  determines  from  moment  to 
moment  the  quantitative  relations  of  the  product  to  the  time.  Now 
in  the  complex  of  events  which  constitutes  growth  not  a  single  sig- 
nificant transformation  is  independent  of  the  rest;  each  must  evidently 
use  some  product  of  other  transformations  and  contribute  some  product 
to  get  another  series  of  processes.  We  can  therefore  understand  how 
the  whole  phenomenon,  notwithstanding  its  complexity  and  the  multi- 
plicity of  the  chemical  reactions  involved  in  it,  may  nevertheless  be 
governed,  as  to  its  quantitative  outcome,  by  the  rate  at  which  a  single 
reaction  occurs.  This  reaction,  as  we  have  seen,  is  autocatalytic. 

We  are  thus  led  to  inquire  whether  the  growth-diagram,  which  is  so 
similar  in  form  to  the  curve  which  represents  the  progress  of  an  auto- 
catalyzed  chemical  reaction,  may  properly  be  regarded  as  establishing 
the  existence  of  Catalyzers  of  Growth  which  are  numbered  among  the 
products  of  the  growth-process,  or  Endogenous  Catalyzers,  as  Hopkins 
has  termed  them,  and  also  the  existence  of  Impeding  Factors,  attribut- 
able either  to  the  exhaustion  of  an  essential  constituent  of  the  reaction, 
or  to  the  accumulation  of  growth-products. 

The  problem  becomes  somewhat  clearer  when  we  consider  the  simple 
case  of  Bacteria,  growing  on  a  limited  amount  of  a  given  culture-medium. 
In  this  case,  as  McKendrick  has  shown,  precisely  analogous  phenomena 
are  exhibited  to  those  which  characterize  the  growth  of  higher  organ- 
isms. The  growth  of  the  bacterial  culture,  measured  by  the  total 
mass  or  number  of  bacteria  produced  at  given  time-intervals,  is  at 
first  extremely  slow;  it  increases  in  velocity,  however,  and  at  first 
almost  in  proportion  to  the  number  of  bacteria  produced.  At  a  later 
stage  growth  is  impeded  and  finally  comes  to  a  standstill  when  the 
density  of  the  population  of  the  culture-medium  has  attained  a  certain 
maximum. 

These  phenomena  are  interpreted  by  McKendrick  in  the  following 
manner:  Each  bacterium  is  capable  of  giving  rise  to  a  certain  number 
of  daughter-cells  in  a  certain  interval  of  time  under  constant  nutritive 
conditions.  This  potentiality  is  transmitted  to  its  offspring,  so  that 
were  the  nutritive  constituents  of  the  culture-medium  inexhaustible, 
the  velocity  of  reproduction  would  always  be  proportionate  to  the 
number  of  bacteria  previously  produced,  or,  in  other  words,  the  density 
of  the  bacterial  population  would  increase  in  geometrical,  while  the 
time  increased  in  arithmetical  progression.  In  practice,  however,  the 
ability  of  the  culture-medium  to  supply  nutritive  materials  to  the 


GENERAL  CHARACTERISTICS  OF  GROWTH-PROCESS      479 

bacteria  is  limited,  and  the  rate  of  multiplication  is  slowed.  McKen- 
drick  infers,  therefore,  that  the  rate  of  multiplication  is  proportional  to 
two  factors;  in  the  first  place  to  the  number  of  bacteria  previously 
produced,  and  in  the  second  to  the  concentration  of  the  still-available 
foodstuffs.  This  leads  to  the  equation: 

dx 

——     =     kx(a    —  x) 
at 

where  "x"  is  the  number  of  bacteria  per  unit-volume,  "a  — x"  is  pro- 
portional to  the  concentration  of  available  nutrients  and  "k"  is  a 
constant  proportionality-factor.  Integration  of  this  differential  equa- 
tion leads  to  the  relationship 

log =     ka(t    -  ti) 

a    —  x 

where  "x"  is  the  number  of  bacteria  per  unit  volume,  a  is  the  maximal 
density  of  population  which  is  attainable  in  a  given  culture  medium, 
"k"  is  a  constant  proportionality-factor  and  ti  is  the  time  at  which 
the  density  of  the  bacterial  population  has  attained  half  its  maximum.1 

The  relationship  between  the  number  or  mass  of  bacteria  produced 
and  the  time  of  incubation  which  is  expressed  in  these  equations  is, 
however,  identical  with  that  which  expresses  the  relationship  between 
weight  and  age  in  any  given  growth-cycle  of  an  animal  or  plant.  It  is 
also  identical  with  the  relationship  between  the  mass  of  the  products 
and  the  time  in  autocatalyzed  chemical  reactions,  such  as  the  hydrol- 
ysis of  Methyl  Acetate.  The  question  therefore  presents  itself,  whether 
the  process  of  growth  in  a  multicellular  organism  such  as  a  mammal  is 
comparable  to  an  autocatalyzed  chemical  reaction,  or  whether  McKen- 
drick's  interpretation  of  the  growth-curve  of  a  bacterial  population  does 
not  offer  an  alternative  explanation  of  the  facts.  In  other  words  two 
alternative  possibilities  would  appear  to  exist :  the  one  that  the  accelera- 
tive  factor  in  growth  is  a  chemical  substance,  as  it  is  in  autocatalyzed 
chemical  reactions,  the  other  that  it  is  simply  due  to  the  multiplication 
of  cells,  each  of  which  is  possessed  of  like  potentialities  of  reproduction. 

On  closer  analysis  it  will  be  seen,  however,  that  these  interpretations,  • 
at  first  sight  alternative,  are  in  reality  identical. 

Reverting  to  the  case  afforded  by  the  multiplication  of  bacteria  in  a 
limited  amount  of  culture-medium,  and  looking  to  the  beginning  and 
end  of  the  process,  we  see  that  the  increase  in  bacterial  population 
means  essentially  that  the  simple,  unorganized  constitutents  of  the 
culture-medium  have  been  transformed  into  the  substances  composing 
the  bacteria.  Any  acceleration  experienced  by  the  process  must 
ultimately  be  due  to  the  preceding  synthesis,  irrespective  of  the  fact 
that  the  synthesis  takes  place  in  a  heterogeneous  system,  i.  e.,  in  the 
separate  particulate  masses  which  form  the  individual  bacteria.  When 

1  I  have  slightly,  but  unessentially,  modified  McKendrick's  formulation  of  this 
relationship  in  order  to  make  clearer  the  analogies  which  follow. 


480  PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

we  say  that  each  bacterium  has  a  like  potentiality  of  reproduction  we 
clearly  express  the  fact  that  the  synthesis  of  bacterial  cell-substances 
which  results  in  the  production  of  a  cell  is  a  favoring  condition  for  the 
production  of  new  cells,  in  other  words  that  some  substance  or  sub- 
stances comprising  the  bacterium  accelerate  the  production  of  new 
masses  of  bacterial  substance.  In  ultimate  terms,  therefore,  the  two 
interpretations  of  the  phenomenon  are  identical,  the  only  essential 
difference  between  the  more  familiar  cases  of  autocatalysis,  such  as  the 
hydrolysis  of  methyl  acetate,  and  the  process  of  cell-multiplication, 
being  the  fact  that  in  the  latter  process  the  reaction  takes  place  in  a 
heterogeneous  chemical  system,  i.  e.,  within  the  particulate  masses 
comprising  the  cells.  Yet  the  fact  that  a  chemical  reaction  takes  place 
in  a  heterogeneous  medium  does  not  imply  that  it  is  discontinuous.  The 
production  of  calcium  sulphate  from  a  mixture  of  calcium  hydrate  and 
sulphuric  acid  is  a  continuous  process  despite  the  fact  that  the  product 
is  divided  into  particulate  masses,  which  in  this  instance  are  crystals. 
On  the  other  hand  the  instances  of  autocatalysis  in  heterogeneous 
systems  are  abundant  in  chemical  literature,  the  oxidation  of  metals 
in  contact  with  air  being  a  familiar  illustration  of  a  group  of  autocata- 
lyzed  reactions  of  this  type. 

The  Accelerative  Factor  in  the  process  of  growth  is,  therefore,  a 
chemical  substance  or  substances,  or  a  chemical  condition,  which  is 
strictly  analogous  to  the  accelerative  factor  in  less  complex  auto- 
catalyzed  phenomena.  The  autocatalytic  character  of  the  growth- 
process  follows  of  necessity,  in  fact,  from  the  fundamental  characteristic 
which,  more  than  any  other,  distinguishes  living  from  non-living  mate- 
rial, namely  its  potentiality  of  unlimited  reproduction.  When  we  assert 
that  living  cells  all  possess  like  potentiality  of  reproduction  we  merely 
state  in  morphological  terminology  that  the  production  of  living  matter 
is  a  self-sustained  or  autocatalyzed  phenomenon.  Just  as  the  produc- 
tion of  living  from  inanimate  matter  is  essentially  a  chemical  process, 
so  the  acceleration  of  its  production  which  is  consequent  upon  the  multi- 
plication of  the  particulate  resultants  of  the  process  is,  when  viewed 
from  the  chemical  standpoint,  evidence  that  substances  are  produced 
in  the  creation  of  living  matter  which  have  the  essential  property  of 
catalyzing  its  further  manufacture.  Regarding  the  possible  nature  of 
these  endogenous  catalyzers,  we  shall  have  something  to  say  in  a  later 
part  of  this  chapter. 

It  remains  to  consider  what  may  be  the  probable  nature  of  the 
Inhibitive  Factor  which  ultimately  brings  the  process  of  growth  to  a 
standstill,  which  sets  a  limit  to  the  normal  dimensions  of  any  given 
species  of  animal,  and  which  predominates  over  the  accelerative  factor 
during  the  latter  half  of  each  growth-cycle.  In  the  simpler  instances 
of  autocatalysis,  as  we  have  seen,  the  inhibitive  factor  may  be,  either 
the  exhaustion  of  the  materials  undergoing  transformation,  or,  on  the 
other  hand,  the  accumulation  and  consequent  "  back-pressure"  of  the 
products  of  the  reaction,  or  both  of  these  factors  may  play  a  part  in 


GENERAL  CHARACTERISTICS  OF  GROWTH-PROCESS       481 

determining  the  magnitude  of  the  inhibition.  Either  of  these  alter- 
natives would  yield  the  time-relations  expressed  in  the  autocatalyzed 
reaction-formula,  for  the  following  reasons : 

In  case  the  velocity  of  the  reverse  reaction  is,  at  all  stages  of  the 
transformation,  negligible  in  comparison  with  that  of  the  forward  reac- 
tion, then  the  only  inhibitive  factor  must  be  the  exhaustion  of  the 
Substrate,  or  material  undergoing  transformation.  The  velocity  of  the 
process  will  be,  as  usual  in  chemical  reactions,  proportional  to  the  mass 
of  untransformed  material  and  also  to  the  mass  of  the  catalyzer,  that  is, 
in  these  instances,  to  the  mass  of  the  products  of  the  reactions.  Desig- 
nating the  mass  of  a  product  of  the  reaction  at  any  moment  by  "x," 
and  "a"  the  initial  amount  of  the  material  undergoing  transformation, 
this  yields  the  relation : 

dx 

Velocity  of  transformation      =     =     kx(a   —  x) 

dt 

which  is  the  formula  characteristic  of  an  autocatalyzed  reaction. 

Coming,  now,  to  the  case  in  which  the  velocity  of  the  reverse  reaction 
is  so  considerable  as  to  be  comparable  with  that  of  the  forward  reaction, 
we  will  assume,  in  the  first  instance,  that  the  materials  undergoing 
transformation  (or  foodstuffs  in  growth)  are  inexhaustible,  i.  e.,  are 
constantly  being  renewed  from  the  environment,  so  that  the  mass  of 
material  undergoing  transformation  is  a  constant  which  we  may 
designate  by  the  symbol  of  "A."  The  velocity  of  the  forward  reaction 
will  then  be,  as  in  the  above  instance,  proportionate  to  the  mass  of  the 
catalyzer  (  =  product  of  the  reaction,  =  "x")  and  also  to  the  constant 
mass  of  substrate,  that  is,  to  "A."  The  velocity  of  the  reverse  reaction 
(breaking-down  of  the  products  of  the  reaction  into  the  initial  sub- 
stances again)  will  be  proportional  to  the  mass  of  the  products  (  =  "x"), 
but  also  to  the  mass  of  the  catalyzer  (  =  "x"),  because  in  the  majority 
of  instances  of  "typical"  catalysis  the  catalyzer  accelerates  both  the 
forward  and  the  reverse  reactions  in  equal  proportion.  The  velocity 
of  the  reverse  reaction  at  any  moment  will  therefore  be  proportionate 
to  x2,  and  the  net  velocity  of  the  process,  being  the  difference  between 
the  velocities  of  the  forward  and  the  reverse  reactions,  will  be  given  by : 

dx 
-      =     kl*A      =     UP 

in  which  "ki"  and  "k2"  are  the  velocity-proportionality  factors  of  the 
forward  and  reverse  reactions  respectively.  Rearranging  the  terms  of 
the  equation  this  may  be  written: 

-^          k 
dt 

which  is  again  identical  with  the  ordinary  formula  of  autocatalysis, 
with  the  exception  that  the  constant  "a,"  denoting  the  maximal  attain- 
able value  of  "x"  is  now  not  the  initial  mass  of  material  undergoing 
31 


482  PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

transformation,  but  the  initial  mass  multiplied  by  the  constant  ratio 
of  the  velocity-constants  of  the  forward  and  reverse  reactions. 

In  the  case  of  the  growth  of  Bacteria  in  a  limited  quantity  of  culture 
medium,  McKendrick  assumes  that  the  inhibitive  factor  is  simply  the 
exhaustion  of  available  foodstuffs,  i.  e.,  that  it  corresponds  to  the  first 
of  the  alternative  possibilities  outlined  above.  In  the  growth  of 
animals,  however,  it  is  difficult  to  see  how  the  limited  availability  of 
Foodstuffs  could  be  a  deciding  factor  in  the  inhibition  of  normal  growth, 
for  the  medium  in  which  our  cells  actually  live  and  grow  is  the  lymph 
(or  "tissue-fluid"),  which  is  constantly  supplied  and  renewed  from  the 
blood.  Now  the  mechanisms  of  the  body  are,  as  we  have  seen,  so 
devised  that  the  composition  of  the  blood  is  maintained  in  a  condition 
of  extraordinary  uniformity.  It  is  true  that  its  content  of  the  more 
particularly  nutritional  constituents  fluctuates  with  the  fluctuating 
absorption  of  nutrients  from  the  alimentary  canal,  but  these  short- 
period  fluctuations  result  in  the  long  run  in  the  mairtenance  of  a 
remarkably  steady  flow  of  nutrient  materials  to  the  tissues.  The  blood 
derives  its  nutrient  constituents  from  the  external  environment  and  in 
fact  contains  them  not  merely  in  sufficient  proportion  to  maintain  an 
equilibrium  of  body-weight,  but,  even  in  adult  animals,  in  considerable 
excess  of  the  necessary  minimum,  the  destruction  of  this  excess  consti- 
tuting the  "Exogenous  Metabolism"  as  contrasted  with  "Endogenous 
Metabolism,"  or  irreducible  minimum  of  nutrient-consumption  incident 
to  the  maintenance  of  life.  The  medium  in  which  our  cells  live,  there- 
fore, is  under  normal  dietetic  conditions  a  medium  of  almost  constant 
composition  and,  for  the  purposes  of  tissue-synthesis,  it  is  inexhaustible 
since  it  is  continually  renewed.  The  Substrates  of  growth  must  there- 
fore be  regarded  as  being  of  constant  concentration  and  the  inhibiting 
factor  of  growth  must  be  sought  elsewhere  than  in  the  exhaustion  of 
available  nutrients. 

On  the  other  hand,  if  a  portion  of  the  tissues  of  an  adult  animal  be 
injured  or  destroyed,  the  process  of  growth  immediately  recommences 
and  is  expressed  in  the  phenomenon  of  Regeneration  which,  if  mechanical 
factors  do  not  impose  an  insuperable  obstacle,  continues  until  the 
complete  restoration  of  the  lost  tissues  has  been  accomplished.  In 
other  words,  removal  of  the  products  of  growth  immediately  reinaugu- 
rates  the  growth-process,  just  as  the  removal  of  the  products  of  a 
"balanced"  chemical  reaction  at  equilibrium  immediately  reinitiates 
the  forward  reaction.  We  must  infer,  therefore,  that  in  the  growth  of 
mammals,  at  least,  it  is  the  accumulation  of  the  Products  of  Growth 
which  normally  inhibits  the  process  and  not  the  exhaustion  of  nutritive 
materials.  In  Plants  the  supply  of  nutritive  materials  to  the  cells  is 
more  fluctuating  and  dependent  upon  the  environment,  and  here  we 
may  expect  to  find,  and  do  actually  find,  a  much  more  conspicuous  part 
played  by  the  supply  of  nutrients  in  determining  the  final  attainable 
dimensions  of  the  organism.  Nevertheless  plants  of  a  given  species, 
even  under  the  most  favorable  nutritional  conditions,  do  not  exceed 
certain  definable  limits  in  their  dimensions  at  maturity,  and  they 


GENERAL  CHARACTERISTICS  OF  GROWTH-PROCESS       483 

i 

display  regeneration  when  portions  of  their  tissues  are  removed. 
Even  in  the  case  of  bacteria  growing  in  a  limited  supply  of  culture- 
medium,  there  is  evidence  which  tends  to  show  that  in  many  cases  the 
accumulation  of  bacteria  or  bacterial  products  really  sets  the  limit  to 
their  multiplication  rather  than  the  exhaustion  of  the  nutrients  in  their 
culture  medium. 

We  infer,  therefore,  that  the  process  of  growth  is  governed  by  a 
series  (in  mammals  usually  three)  of  autocatalyzed  chemical  reactions 
in  which  the  factor  which  determines  the  retardation  and  ultimate 
equilibrium  of  the  process  is  the  accumulation  of  the  products,  i.  e., 
the  growth  itself. 

The  constancy  of  the  concentration  of  Growth-substrates  in  animals 
affords  a  readily  intelligible  explanation  of  the  extraordinary  simplicity 
of  the  quantitative  relationship  between  growth  and  time,  which,  as 
we  have  seen,  so  frequently  obtains.  The  relationship  in  question  is 
that  which  characterises  the  progress  of  an  autocatalyzed  monomolecu- 
lar  reaction,  and  even  admitting  the  probability  that  a  single  chemical 
transformation  may  determine  the  speed  and  set  the  pace  for  the 
whole  of  the  multitudinous  variety  of  chemical  processes  involved  in  the 
growth  of  new  protoplasm,  yet  it  may  seem  strange  that  even  this 
single  reaction  should  be  of  so  simple  a  character,  more  especially  since,, 
as  the  construction  of  protoplasm  involves  synthesis  of  large  out  of 
relatively  small  molecules,  we  would  expect  any  reaction  involved  in 
growth  to  be  multimolecular.  Now  this  may  actually  be  the  case,  even 
in  the  Master-reaction  which  determines  the  quantitative  outcome  of 
all  the  growth-processes,  for  if  the  concentration  of  the  substrates  of 
growth  remains  undiminished  by  the  growth  which  occurs,  then  any 
number  of  molecules  of  the  substrates  may  participate  in  the  synthesis 
which  constitutes  the  governing  reaction,  without  involving  any  depar- 
ture of  the  relationship  between  the  time  and  extent  of  growth  from 
that  which  is  expressed  in  the  monomolecular  autocatalytic  formula. 
If  "n"  molecules  of  the  substrate  combine  to  form  one  molecule  of  the 
product,  then  the  velocity  of  the  forward  reaction  will  be  given  by:  ' 


while  that  of  the  backward  reaction  will  be  given,  as  before,  by 

*L     _    k2x* 
dt 

hence  the  net  effect,  or  actual  growth,  will  be  given  by 

dx  k  xAn  _  k  x2 

"dT 

which,  rearranging  the  terms,  becomes: 

dx 
=     k 

dt 


484     PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

which  is  again  of  the  monomolecular  form,  save  that  the  constant  "a" 
in  the  formula  is  no  longer  proportional  to  the  actual  concentration 
of  the  substrates,  but  to  the  nth  power  of  their  concentration.  That 
the  backward  reaction  should  be  monomolecular  is,  of  course,  not  a 
matter  for  surprise,  since  we  may  suppose  that  the  majority  of  decom- 
positions which  living  tissue  suffers  consists  in  the  interaction  of  a  single 
molecule  of  some  protoplasmic  constituent  either  with  water  or  with 
oxygen,  the  concentration  of  both  of  which  substances  is  maintained 
automatically  at  an  approximately  constant  level  in  the  tissues. 

Thus  the  synthesis  of  a  protein  involves  the  interaction  of  many 
different  amino-acid  molecules,  but  its  hydrolysis  in  dilute  aqueous 
solution  obeys  the  monomolecular  formula,  because  only  a  single  species 
of  molecule,  that  of  the  protein  itself,  is  undergoing  appreciable  change 
of  mass  or  concentration  in  the  process. 

Summarizing  the  general  characteristics  of  the  growth-process  we 
may  therefore  state : 

1.  That  the  growth  of  man  and  of  animals  takes  place  in  periods  or 
cycles  in  which  slow  and  rapid  growth  alternate,  three  of  the  cycles 
being  usually  appreciable  in  magnitude. 

2.  Each  of  the  growth-cycles  is  the  expression  of  an  underlying  self- 
accelerated  chemical  process. 

3.  The  accelerating  factor  is  some  substance  or  group  of  substances 
produced  during  growth. 

4.  The  supply  of  nutriment  capable  of  transformation  into  living 
tissues  may,  in  normal  animals,  be  regarded  as  constant  and  undimin- 
ished  by  the  process  of  growth  itself. 

5.  The  inhibiting  factor,  which  ultimately  brings  the  growth  in  any 
given  cycle  to  a  standstill,  is  the  accumulation  of  the  products  of  growth. 

6.  Removal  of  these  products,  as  by  local  death  or  injury,  or  by 
general  inanition,  reinaugurates  the  process  of  growth,  which  continues 
until  equilibrium  is  reattained. 

7.  The  whole  of  the  diverse  processes  which  in  the  aggregate  con- 
stitute growth  are  governed  and  determined  in  rate  and  magnitude  by 
the  specificially  slowest  essential  process. 

8.  The  forward  reaction  in  the  governing  process  may  involve  the 
interaction  of  many  different  molecules,   but  the  reverse  reaction 
appears,  in  many  cases  at  least,  to  involve  the  decomposition  of  only 
a  single  species  of  molecule  of  variable  mass  or  concentration. 


THE  INFLUENCE  OF  RACE,  SEX,  AND  ENVIRONMENT  UPON 
THE  GROWTH-PROCESS. 

The  fact  that  the  bodily  dimensions  of  a  given  species  of  animal 
never  exceed  certain  characteristic  upper  limits,  no  matter  how  favor- 
able the  environmental  conditions  may  be  with  respect  to  the  abun- 
dance and  variety  of  Nutrients,  shows  that  the  factors  which  inhibit  the 


INFLUENCE  OF  RACE  AND  SEX  ON  GROWTH-PROCESS    485 

growth  in  any  given  growth-cycle  are  primarily  characteristic  of  the 
process  itself  and  only  in  a  minor  degree  dependent  upon  the  dietary, 
provided  it  is  in  all  respects  sufficient.  We  have  seen  that  the  main 
inhibiting  factor  in  growth  arises  from  the  accumulation  of  the  products 
of  growth  and  the  enhanced  rapidity  of  tissue-disintegration  which 
ensues.  The  characteristic  dimensions  of  an  animal,  therefore,  and 
the  same,  to  a  less  striking  degree,  is  doubtless  true  of  a  plant,  are 
determined  mainly  by  the  relative  magnitude  of  the  specific  Velocity- 
constants  of  the  forward  and  the  opposed  reactions.  These  are  char- 
acteristic of  the  particular  reactions  which  occur  in  a  given  race  or 
sex,  and  are  not  influenced  by  the  mere  abundance  or  paucity  of  the 
dietary. 

That  the  bodily  dimensions  of  an  animal  may  be  affected  to  a  limited 
extent  by  the  abundance  of  the  Dietary  is,  however,  a  readily  ascer- 
tainable  fact.  If  the  dietary  be  absolutely  insufficient  even  to  main- 
tain bodily  heat  and  the  output  of  work,  the  tissues  are  called  upon 
to  supply  the  energy-requirements,  the  animal  loses  weight  and  may 
ultimately  die  of  inanition  or  of  acute  conditions  supervening  upon 
partial  inanition.  If  the  dietary  insufficiency  is  less  extreme  than  this, 
growth  is  nevertheless  slowed,  and  the  bodily  dimensions  attainable 
at  maturity  are  smaller  than  is  normal  for  the  species.  If,  on  the  other 
hand,  the  diet  is  exceedingly  abundant  and  other  environmental 
conditions  are  exceptionally  favorable,  then  the  bodily  dimensions 
at  maturity  may  come  to  distinctly  exceed  the  average,  although  the 
degree  of  supernormality  which  is  attainable  in  this  way  is,  of  course, 
strictly  limited.  Mice,  under  no  matter  what  favorable  conditions  of 
environment  and  abundance  of  food  supplies,  do  not  achieve  the  bodily 
dimensions  of  a  guinea-pig  or  even  of  a  rat. 

The  supply  of  nutrients  to  the  tissues  is,  as  we  have  seen,  determined 
primarily  by  the  composition  of  the  blood  which,  subject  to  short- 
period  fluctuations,  remains  relatively  constant  throughout  the 
growth  and  life  of  the  animal.  The  "Nutrient  Level"  or  concentration 
of  growth-substrates  in  the  blood  is  maintained  by  a  dynamic  equilib- 
rium which  involves  a  variety  of  factors.  On  the  one  hand  we  have  the 
availability  of  Foodstuffs  in  the  external  environment  and  the  ability 
of  the  digestive  apparatus  to  disintegrate  them  and  to  absorb  the 
products  of  their  disintegration.  On  the  other  hand  we  have  the  rate 
of  utilization  by  the  tissues  and  the  equilibrium  between  the  storage- 
capacities  of  the  tissues  for  the  various  classes  of  foodstuffs,  for  poly- 
saccharides,  fats,  and  amino-acids,  and  the  concentration  of  these 
substances  or  their  products  in  the  blood  and  tissue-fluids.  The  height 
of  the  nutrient  reservoir  in  the  blood  is  thus  governed  by  a  balance 
between  a  certain  rate  of  inflow  and  a  certain  rate  of  outflow.  In 
addition  to  these  factors,  and  in  order  to  avoid  an  excessive  accumula- 
tion of  nutrient  materials  in  the  blood,  an  overflow  is  also  provided  in 
the  phenomenon  of  Exogenous  Metabolism,  or  the  destruction  of  food- 


486    PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

stuffs  which  have  not  yet  come  to  comprise  living  matter,  a  process 
which,  in  the  case  of  the  amino-acids  at  all  events,  forms  a  very  large 
proportion  of  the  total  metabolism  of  a  normally  nourished  animal. 

If  any  of  these  several  factors  is  decidedly  altered  in  magnitude  or 
velocity  a  more  or  less  marked  effect  upon  bodily  weight  will  ensue. 
Thus  if  the  rate  of  inflow  be  diminished  beyond  a  certain  point  by  an 
insufficient  dietary  the  nutrient  level  sinks  and  growth  is  retarded,  or, 
in  the  adult  animal  which  has  attained  growth-equilibrium,  the  process 
of  growth  may  be  reversed  and  loss  of  tissue  occur.  The  extent  of  this 
reversion  is  strictly  limited  in  the  more  complex  forms  of  metazoa  by 
the  necessity  of  maintaining  certain  mechanical  conditions :  the  integrity 
of  the  skeleton,  the  functional  ability  of  the  digestive  organs,  the  pulsa- 
tion of  the  heart,  the  integrity  of  a  closed  vascular  system,  the  coordi- 
nating activities  of  the  nervous  system,  and  the  continuance  of  respira- 
tory movements.  If  any  of  these  suffer  in  so  complex  an  organization 
as  our  own  the  whole  must  fail  and  death  ensue.  But  in  some  less 
complex  forms,  as  in  the  fresh  water  worm  Planaria,  starvation  actually 
accomplishes  Reversion  of  Growth  until  an  embryonic  stage  of  develop- 
ment is  regained  (Child). 

If,  on  the  other  hand,  the  rate  of  inflow  of  nutrients  be  maintained 
unaltered  and  the  rate  of  outflow  increased  or  diminished  the  rate  of 
accretion  of  tissue  must  obviously  be  affected  to  a  proportionate  degree. 
In  normal  cases,  since  the  rate  of  outflow  or  consumption  of  nutrients 
for  tissue-building  purposes  is  determined  by  the  relative  magnitudes 
of  the  specific  velocity-constants  of  upbuilding  and  disintegration,  the 
rate  of  outflow  will  vary  in  different  species  and  not  improbably  in  the 
two  sexes  of  the  same  species,  and  to  a  certain  extent  in  different 
individuals.  The  environment,  on  the  contrary,  provided  the  inflow 
of  nutrients  is  maximal  or  at  least  sufficient,  may  be  expected  to  play 
little  if  any  part  in  determining  the  rate  of  outflow. 

The  rate  of  overflow  is  also  conditioned  primarily  by  internal  regula- 
tion, but  we  may  observe  the  effects  of  its  alteration  in  so  far  as  the 
nutrient-level  of  the  amino-acids  is  concerned,  by  the  pronounced  effects 
of  hyper-  or  hypo-activity  of  the  Thyroid  upon  the  development  of  the 
tissues.  The  administration  of  thyroid  extract  leads  to  a  very  decisive 
increase  in  the  rate  of  Deaminization  of  amino-acids,  and  in  normal 
adults  who  have  attained  growth-equilibrium,  this,  which  involves  a 
fall  of  the  nutrient-level,  results  in  progressive  loss  of  weight  which 
may,  if  it  affects  essential  tissues,  result  in  dangerous  or  even  fatal 
symptoms.  The  effects  of  hypo-activity  are  the  opposite  and  the 
excessive  accretions  of  tissue  not  being  uniformly  distributed,  aberra- 
tions of  growth  occur  which  culminate  in  the  condition  of  Myxedema. 
In  amphibians  excision  of  the  thyroid,  as  Gudernatsch  has  very 
strikingly  demonstrated,  results  in  the  arrest  of  Metamorphosis, 
possibly  because  the  degeneration  of  certain  tissues  which  is  a  necessary 
precedent  of  metamorphosis  cannot  occur. 


INFLUENCE  OF  RACE  AND  SEX  ON  GHOWTtt-PliOCESS    487 
In  the  autocatalytic  formula  as  applied  to  the  process  of  growth: 

log =     ka(t    -  ti) 

a   —  x 

the  constant  "a"  is  proportional  to  some  exponent  of  the  concentration 
of  growth-substrates,  i.  e.,  to  the  Nutrient-level.  In  any  given  species, 
therefore,  we  may  expect  to  find  that  within  certain  limits  its  magnitude 
is  affected  by  the  environment  and  especially  by  the  abundance  or 
paucity  of  the  dietary.  The  constant  "  k"  on  the  contrary  is  expressive 
of  the  specific  velocity  of  the  process  of  tissue-disintegration,  charac- 
teristic of  the  species,,  probably  of  the  sex,  and  peculiar  even  to  a 
particular  individual.  Thus  we  may  expect,  in  a  given  species,  to  find 
that  its  magnitude  is  unaffected  by  the  environment,  but  dependent 
upon  Sex  and  Race.  We  have  seen  that  the  autocatalytic  formula 
applies  to  the  first  nine  months  of  extra-uterine  growth  in  infants  and 
that  the  values  of  "a"  and  "k"  may  be  computed  from  all  of  the 
observed  weights  at  the  various  ages  chosen  for  the  comparison  of  the 
equation  with  the  results  of  actual  measurement.  In  the  following 
table  the  values  of  "a"  and  "k"  for  British  Infants  born  in  England 
and  in  Australia  respectively  and  for  South  German  infants  born  in 
Frankfurt  (from  the  data  of  Schmidt-Monnard)  are  compared: 

COMPARISON  OF  THE  EFFECTS  OF  RACE  AND  ENVIRONMENT  UPON  THE 
PARAMETERS  OF  THE  GROWTH-CURVE. 

Males.  Females. 

Race  and  place  of  birth.  a  (ounces).         kxlO6.  a  (ounces).          kxlO6. 

British  (born  in  England)      .      .      318  399  312  340 

British  (born  in  Australia)     .      :      341.5  398  350  317 

South  German       .....     315  451  290  537 

It  will  be  seen  that  the  parameters  or  constants  of  the  growth  curve 
of  infants  are  affected  in  the  sense  indicated  by  the  above  discussion 
by  the  factors  of  sex,  race  and  environment.  While  the  value  of  "a" 
is  not  greatly  affected  by  sex  or  by  dissimilarity  of  race,  the  values 
obtained  in  the  similar  environments  of  Frankfurt  and  London  being 
very  alike,  it  is  greatly  affected  by  dissimilarities  in  environment,  as  a 
comparison  of  the  values  of  "  a"  in  Australia  and  in  Europe  shows.  On 
the  other  hand,  "k"  is  comparatively  unaffected  by  environment,  being 
practically  identical  for  British  males,  whether  born  in  Australia  or  in 
England,  and  very  nearly  the  same  for  British  females  born  in  these  two 
environments,  whereas  it  is  profoundly  affected  in  magnitude  by  sex 
and  race,  as  indicated  by  the  marked  difference  in  the  values  of  "k" 
for  males  and  females  and  for  South-German  as  compared  with  British 
infants. 

When  it  is  remembered  that  these  parameters  have  not  been  calcu- 
lated arbitrarily,  but  that  they  are  computed  by  the  method  of  least 
squares  from  all  of  the  observations  and  therefore  partake  in  some 
measure  in  the  errors  incident  to  the  observations,  it  will  be  seen  that 


488    PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

the  above  data  afford  a  very  remarkable  demonstration  of  the  correct- 
ness of  the  view  that  growth  is  determined  by  an  underlying  auto- 
catalyzed  chemical  process.  It  is  furthermore  clear  that  the  form  of 
the  curve  of  growth  in  normal  infants  is  determined  by  two  separate 
groups  of  factors.  The  one,  analogous  to  the  absolute  mass  of  the 
reacting  substances  in  a  chemical  reaction,  being  dependent  upon  the 
environment  and  probably  largely  influenced  by  the  abundance  or 
deficiency  of  the  habitual  dietary;  while  the  other,  analogous  to  the 
specific  velocity  of  a  chemical  reaction,  is  relatively,  if  not  absolutely, 
independent  of  environmental  or  nutritional  conditions,  and,  being 
expressive  of  the  nature  of  the  growth-process  itself  as  distinguished 
from  the  availability  of  the  materials  for  growth,  is  distinctively  modi- 
fied by  race  and  sex. 


THE  SUBSTRATES  OF  GROWTH. 

The  substrates  of  growth,  i.  e.,  the  material  out  of  which  living  tissues 
are  synthesized,  are  the  Foodstuffs,  namely  oxygen,  water,  inorganic 
salts,  carbohydrates,  fats  and  proteins.  In  the  period  of  biochemical 
research  which  immediately  followed  the  fundamental  discoveries  of 
Liebig  and  Voit,  the  application  of  the  laws  of  the  conservation  of 
matter  and  energy  to  the  phenomena  of  growth  and  metabolism 
appeared  to  supply  all  of  the  necessary  clues  for  the  interpretation  of 
the  relationship  of  the  foodstuffs  to  the  maintenance  of  life.  But  with 
the  increasing  refinement  of  our  knowledge  of  the  intimate  chemical 
structure  of  the  foodstuffs  themselves  it  has  become  increasingly  appar- 
ent to  us  during  the  recent  decades  that  it  is  not  sufficient  merely  to 
supply  an  animal  or  a  human  being  with  a  sufficiency  of  nitrogen, 
carbon  and  calories  to  replace  his  daily  waste  in  order  to  maintain  the 
equilibrium  between  waste  and  repair  in  his  tissues,  nor  is  it  even 
sufficient  to  supply  these  desiderata  in  digestible  and  assimilable  form; 
it  is  furthermore  necessary  to  supply  irreducible  minima  of  specified 
atomic  groupings  or  complexes  of  nitrogen,  carbon,  hydrogen  and  so 
forth  which,  it  appears,  are  essential  constituents  of  living  matter, 
and  yet  are  not  synthesizable  by  animal  tissues.  Thus  the  Pyrrole 
grouping,  for  example  (see  Chapter  XV),  which  is  an  essential  building- 
stone  of  Hemoglobin,  would  appear  to  be  as  much  an  elementary 
requirement  of  animals  as  nitrogen  or  carbon  itself,  inasmuch  as, 
according  to  Abderhalden,  they  are  unable  to  synthesize  it  from  other 
carbon  or  nitrogen  complexes  in  the  diet  and,  lacking  it,  are  just  as 
assuredly  suffering  starvation  as  if  they  were  lacking  one  of  the  more 
elementary  desiderata. 

The  variety  of  these  essential  constituents  of  the  diet  with  which  we 
are  acquainted  is  already  very  great  and  is  unquestionably  destined  to 
grow  with  increasing  scope  and  refinement  of  investigation.  It  is 
highly  probable  that  many  of  the  raw  materials  from  which  the  various 


SUBSTRATES  OF  GROWTH  489 

Internal  Secretions  are  synthesized  are  dietary  constituents  of  this 
essential  type,  for  example  the  Iminazolyl-grouping,  which  in  all 
probability  forms  an  essential  constituent  of  the  active  principles  of 
both  lobes  of  the  pituitary  body,  the  Catechol-grouping  which  is  an 
essential  complement  of  the  molecule  of  Adrenalin,  and  the  Indole 
radical  which,  from  the  observations  of  Kendall,  would  appear  to  be  a 
component  of  the  active  principle  of  the  thyroid,  are  examples  which 
will  serve  to  illustrate  the  essential  importance  of  specific  molecular 
groupings  or  arrangements  of  atoms,  which,  if  not  synthesizable  by 
animal  tissues,  must  necessarily  form  a  part  of  the  diet  in  order  to 
maintain  bodily  equilibrium;  and  to  a  still  greater  extent,  of  course,  in 
order  to  render  normal  growth  a  possibility. 

The  Vitamines,  which  appear  to  be  nitrogenous  substances  closely 
related  to  the  Purines,  are  dietary  constituents  of  this  type.  They  are 
essential  for  growth,  and  even  for  the  maintenance  of  bodily  equi- 
librium, yet  the  amount  required  to  maintain  the  weight  of  the  body 
or  to  permit  satisfactory  growth  is  extremely  minute.  They  evidently 
represent  a  group  of  non-synthesizable  essential  constituents  of  living 
matter  which  would  appear  not  to  be  excessively  complicated  in  struc- 
ture since  they  are  usually  obtainable  in  crystalline  form  and  their 
relationship  to  the  pyrimidines  and  the  purines  has  frequently  been 
established. 

Then,  again,  there  are  fatty  constituents  or  substances  soluble  in 
Fats  which  are  probably  of  a  more  complex  character  and  which 
are  equally  essential  elements  of  a  complex  dietary.  According  to  the 
older  view  of  metabolism,  fats  and  carbohydrates  were  considered  to  be 
mutually  replaceable  in  the  dietary  in  isodynamic,  i.  e.,  equicalorific 
proportions.  Provided  the  fats  in  the  dietary  be  not  too  greatly  dimin- 
ished this  is  still  recognized  to  be  true,  but  it  has  now  been  repeatedly 
shown  that  development  and  maintenance  upon  an  absolutely  fat-free 
diet  is  impossible,  no  matter  what  excess  of  carbohydrate  may  be 
furnished  and,  furthermore,  that  Vegetable  Oils  do  not  supply  this 
deficiency.  According  to  McCollum  the  essential  constituents  of  the 
diet,  in  addition  to  the  requisite  mineral  salts,  amino-acids  and  calorific 
value  in  the  form  of  fats  or  carbohydrates,  fall  into  two  groups,  of  which 
one  is  soluble  in  water,  while  the  other  is  insoluble  in  water  and  is 
soluble  in  fats  and  in  fat-solvents  such  as  ether.  It  is  immaterial  from 
what  source  these  constituents  may  be  derived;  provided  merely  that 
they  are  both  present  and  the  diet  conforms  to  the  other  requirements 
outlined  it  will  suffice  to  maintain  life  and  permit  growth.  These 
substances  have  been  provisionally  designated  by  McCollum  "Fat- 
soluble  A"  and  "Water-soluble  B";  we  are  as  yet  ignorant  of  their 
structure  or  affinities.  But  from  their  essentiality  for  growth,  and 
even  maintenance  for  any  prolonged  period,  we  may  infer  that  they  are 
Substrates  or  raw  materials  which  are  required  in  the  manufacture  of 
living  tissue  and  cannot  be  synthesized  by  the  tissues  themselves. 

The  clearest  indication  of  the  dependence  of  tissue-synthesis  upon  the 


490  PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

presence  of  specific  atomic  groupings  in  the  dietary  is,  however,  afforded 
by  the  investigations  of  Hopkins  and  Willcock  and  of  Osborne  and 
Mendel  upon  the  ability  of  various  pure  Proteins  to  supply  the  nitrogen- 
requirements  of  growth  and  maintenance. 

We  have  seen  that  the  various  protein  constituents  of  the  tissues  and 
of  the  diet  are  built  up  out  of  varying  permutations  and  combinations 
of  a  limited  number  (nineteen  in  all)  .of  Amino-acid  radicals  which  are 
linked  together  in  long  chains.  Now  certain  of  these  nineteen  radicals 
are  lacking  in  some  of  the  proteins,  and  the  administration  of  such 
proteins  to  growing  animals  as  the  sole  source  of  nitrogen  in  the  diet 
enables  us  to  ascertain  whether  the  amino-acid  which  is  lacking  is 
synthesizable  by  animal  tissues,  for  synthesized  it  must  be  if  normal 
tissues  are  to  be  produced  by  the  animal  and  it  is  not  procurable 
preformed  in  the  diet. 

From  the  investigations  cited  it  appears  very  probable  that  the 
only  amino-acid  radical  which  is  synthesizable  by  animal  tissues  is 
Glycocoll,  or  Amino-acetic  Acid.  Of  the  remainder,  it  is  probable  that 
all  must  be  present  preformed  in  the  diet  in  order  to  permit  the  accre- 
tion of  living  tissue;  at  all  events  this  has  been  positively  established 
for  several  of  the  amino-acid  radicals,  for  example  Lysine,  Tryptophane, 
Tyrosine,  and  Cystine. 

The  alcohol-soluble  protein  of  maize,  Zein,  is  lacking  in  glycocoll, 
tryptophane  and  lysine,  and  the  investigations  of  Hopkins  and  Willcock 
and  of  Osborne  and  Mendel  have  shown  that  if  Zein  be  the  sole  source 
of  nitrogen  in  the  diet,  not  only  is  accretion  of  fresh  tissue  impossible, 
but  the  maintenance  of  that  already  formed  is  also  impossible,  so  that 
when  supplied  with  abundance  of  nitrogen,  carbon  and  salts  in  correct 
proportion,  water  and  calories,  the  animal  nevertheless  dies  of  inani- 
tion. If  tryptophane  be  added  maintenance  becomes  possible,  but  not 
growth.  On  such  a  diet,  or  if  supplied  with  Gliadin  which  lacks  only 
glycocoll  and  lysine,  a  young  animal  lives  but  ceases  to  grow  and 
maintains  an  infantile  appearance,  and  full  capacity  to  grow  upon 
readmission  of  the  lacking  constituent  to  the  diet,  until  what  would 
normally  be  a  "ripe  old  age"  (Fig.  33).  Upon  addition  of  lysine  as  well 
as  tryptophane,  normal  growth  and  maintenance  are  at  once  rendered 
possible,  the  glycocoll  being  synthesized  by  the  animal  itself.  Evi- 
dently the  Endogenous  Metabolism,  or  waste  incidental  to  and  an  essen- 
tial consequence  of  life,  of  the  amino-acid  lysine  is  reducible  to  zero, 
possibly  because  a  limited  supply  of  lysine  may  be  utilized  over  and  over 
again  in  the  processes  of  waste  and  repair,  while,  on  the  contrary,  the 
endogenous  metabolism  of  tryptophane  is  not  reducible  to  zero,  possibly 
because  it  is  employed,  not  only  in  the  manufacture  of  tissue,  but  also 
of  constituents  of  the  body  which  undergo  irreversible  consumption. 
The  result  is,  at  all  events,  that  an  inevitable  waste  of  tryptophane 
attends  the  maintenance  of  life,  and  in  its  absence  from  the  diet,  the 
tissues  of  animals  being 'unable  to  synthesize  it  from  other  nitrogenous 
constituents  of  the  diet,  tissue-waste  can  no  longer  be  accurately 


SUBSTRATES  OF  GROWTH 


491 


balanced  by  tissue-repair,  and  continuous  loss  of  tissue  on  an  otherwise 
abundant  diet  is  the  inevitable  outcome. 


FIG.  33. — A  and  B  show  the  contrast  between  two  rats  of  the  same  age,  one  of 
which,  B,  has  been  stunted  by  receiving  a  diet  (protein-free  milk  and  gliadin) ,  deficient 
in  lysine.  The  lower  two  pictures  afford  a  comparison  between  two  rats  of  the  same 
weight  but  widely  differing  in  age.  The  older,  stunted  rat,  B,  has  not  lost  the  character- 
istic proportions  of  the  younger  animal,  C.  (After  Under  hill.) 


It  has  long  been  realized  that  Gelatin  is  not  in  itself  an  adequate 
protein  for  the  maintenance  of  nitrogenous  equilibrium,  although  it  is 
a  "  sparer  of  protein/'  i.  e.,  can  furnish  a  portion,  but  not  the  whole  of 
the  nitrogen  in  the  diet.  We  now  recognize  that  this  is  due  to  the 
absence  of  tyrosine  and  tryptophane  from  the  molecule  of  this  protein. 


492  PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

Casein  is  an  inadequate  protein  on  account  of  its  deficient  content  of 
cystine  (Fig.  34).  In  milk  this  deficiency  is  supplied  by  Lactalbumin. 
In  addition  to  the  various  dietary  constituents  which  have  been 
definitely  ascertained  to  be  essential  and  irreplaceable  there  are  others 
which  we  can  infer,  from  known  data,  to  be  equally  essential.  Thus 
Cholesterol  has  been  shown  by  Gardner  and  his  collaborators  not  to  be 
synthesized  by  animal  tissues;  the  cholesterol  in  the  blood  and  tissues 
being  proportionate  to  the  cholesterol  derivable  from  the  dietary. 
Now  cholesterol  is  an  essential  constituent  of  nervous  tissues,  and 
derivatives  of  cholesterol,  such  as  the  bile-acids,  play  an  essential  part 
in  the  bodily  economy.  A  diet  lacking  in  cholesterol,  which  is  not  at 
the  same  time  lacking  in  other  essential  dietary  constituents,  is  very 


-soo 

Mo 
Uo 

IfO 

llo 
too 
no 

11,0 

IVO 

no 

/CO 

to 

to 
to 


Each  division  -  to  day* 


Days 


Each  division  -  20  days 

FIG.  34. — Curves  of  growth  of  rats  on  basal  rations  plus  casein,  showing  effect  of 
addition  of  cystiiie  to  an  inadequate  allowance  of  casein.  (After  Osborne  and  Mendel.) 

difficult  to  devise,  so  that  direct  proof  of  its  essentiality  in  the  dietary 
has  not  yet  been  adduced.  Still  we  may  infer  with  a  fair  degree  of 
confidence  that  a  certain  minimal  content  of  cholesterol  in  the  diet  is 
requisite,  if  not  for  maintenance,  then  at  least  for  normal  growth  and 
development. 

From  these  various  investigations  it  is  clear  that  the  elementary 
substrates  of  growth  are  in  all  probability  very  numerous  and  represent 
a  variety  of  chemical  genera,  or  at  any  rate,  lipoidal  substances,  basic 
substances  and  amino-acids,  superadded  to  the  more  elementary 
requirements  of  nitrogen,  carbon  and  calories. 

It  is  furthermore  evident  that  growth,  like  all  other  chemical  trans- 
formations, is  absolutely  dependent  upon  its  raw  materials  or  sub- 
strates, and  cannot  occur  in  their  absence.  On  the  other  hand,  the 


RELATIONSHIP  OF  ENDOCRINE  ORGANS  TO  GROWTH     493 

capacity  to  grow,  as  the  above-cited  investigations  of  Osborne  and 
Mendel  reveal,  is  not  determined  by  age  but,  as  we  have  already  con- 
cluded upon  other  grounds,  by  a  lack  of  balance  between  the  forward 
and  opposed  reactions  of  tissue-synthesis  and  tissue-degradation,  so 
that  upon  admission  of  the  necessary  substrates,  no  matter  what  the 
age  prior  to  the  death  or  senescence  of  the  animal  may  be,  growth 
occurs  and  continues  until  equilibrium,  or  equality  of  the  velocities  of 
tissue-synthesis  and  tissue-degradation  is  attained.  We  thus  reach 
once  more,  and  from  a  totally  different  angle,  the  conclusion  that 
the  relatively  stationary  weight  of  an  adult  animal  is  determined 
by  the  accumulation  of  the  Products  of  Growth,  and  not  in  any  sense 
by  the  exhaustion  of  its  Substrates. 

THE  RELATIONSHIP  OF  THE  ENDOCRINE  ORGANS  TO 

GROWTH. 

We  have  seen  that  the  chemical  processes  which  underlie  the  growth 
of  animals  are  of  such  a  nature  that  they  produce  their  own  catalyzers. 
But  if  this  be  so  then  we  are  immediately  impelled  to  the  conclusion 
that  Catalyzers  of  Growth  exist,  i.  e.,  substances  which,  perhaps  in 
minute  proportion,  and  certainly  quite  independently  of  their  nutritive 
or  substrate-  value  may  profoundly  modify  the  growth  of  living  tissues. 
The  question  now  arises  whether  any  evidence  other  than  evidence  of 
this  inferential  kind  is  obtainable  of  the  veritable  existence  of  such 
endogenous  catalyzers  of  growth? 

In  the  simpler  undifferentiated  organisms  the  catalysis  of  growth,  in 
common  with  all  the  other  vital  processes,  is  doubtless  a  function  of 
every  cell,  and  each  cell  contains  the  necessary  materials  for  the  accel- 
eration of  the  production  of  living  matter.  In  the  higher  and  more 
differentiated  organisms,  on  the  other  hand,  it  is  not  at  all  improbable 
that  the  function  of  growth-catalysis  is,  to  a  greater  or  less  extent, 
delegated  to  special  cell-groups  or  organs,  just  as  the  function  of  motility 
is  delegated  to  muscle-cells,  that  of  conductivity  is  especially  displayed 
by  nerve-fibers,  and  those  involved  in  digestion  are  delegated  to  the 
alimentary  canal  and  dependent  organs.  We  are  thus  led  to  direct 
our  attention  to  the  possibility  of  the  existence  in  the  body  of  special 
cell-groups  exercising  to  an  exceptional  degree  the  function  of  growth- 
catalysis. 

The  profound  significance  of  certain  of  the  various  Endocrine  Organs 
or  glands  of  internal  secretion  in  the  processes  of  growth  immediately 
suggests  that  these  are  the  special  cell-groups  to  which  the  function  of 
growth-catalysis  is  most  particularly  delegated.  We  know  from 
abundant  clinical  experience  that  disorders  of  the  thyroid,  thymus, 
sexual  glands  and  particularly  of  the  anterior  lobe  of  the  pituitary 
body,  are  reflected  in  a  profoundly  disturbed  development  of  the 
various  tissues  of  the  body,  while  the  action  of  the  secretions  of  the 
Corpora  Lutea  in  stimulating  the  outgrowth  of  placentae  from  the  wall 


494  PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

of  the  uterus  is  a  striking  example  of  the  intensity  and  specificity  of 
growth-stimulation  which  may  be  brought  about  by  agencies  of  this 

type. 

It  is  possible  that  not  all  of  the  organs  of  internal  secretion  which 
are  capable  of  affecting  and  modifying  the  growth  of  animals  do  so  by 
virtue  of  growth-catalyzers  which  they  elaborate.  Thus  hyperactivity 
of  the  thyroid  leads  to  generalized  loss  of  body-weight  owing  to  a 
marked  increase  of  metabolism  and  particularly  of  nitrogenous 
metabolism,  while  hypo-activity  leads  to  the  peculiar  maladjustments 
of  development  which  characterize  the  condition  of  myxedema.  These 
effects,  however,  are  more  probably  due  to  a  general  action  of  the 
thyroid  principle  in  accelerating  Exogenous  Metabolism  and  reducing 
the  nutritional  level  in  the  tissue-fluids.  They  are  effects  which  more 
probably  concern  the  concentration  of  the  available  substrates  of  growth 
than  the  specific  rapidity  of  their  elaboration  into  protoplasm.  The 
disproportionate  growth  of  connective  tissues  which  characterizes 
myxedema  is  more  probably  to  be  attributed  to  the  absence  of  the 
normal  competition  with  the  cellular  elements  for  a  limited  supply 
of  substrates  than  to  any  specific  stimulation '  of  connective-tissue 
synthesis. 

The  function  of  the  Thymus  in  growth  is  obscure  and  its  true  signifi- 
cance may  perhaps  be  rather  that  of  a  storehouse  of  substances,  for 
example  Nucleic  Acids,  which  will  be  required  in  subsequent  develop- 
ment than  of  a  factory  of  growth-catalyzers.  The  relationship  of  the 
anterior  lobe  of  the  Pituitary  Body  to  the  processes  of  growth  is,  how- 
ever, clearer  and  more  defined,  and  is  of  such  a  character  as  to  en- 
courage the  supposition  that  in  the  hypophysis  we  have  one  instance 
among  others  of  an  organ  in  which  the  function  of  growth-catalysis  is 
concentrated  and  specialized. 

The  relationship  of  the  pituitary  gland  to  certain  remarkable  disturb- 
ances of  growth  was  first  pointed  out  in  1888  by  the  French  surgeon 
Pierre  Marie,  who  drew  attention  to  two  types  of  anomalous  growth 
which  postmortem  examination  showed  to  be  invariably  associated 
with  abnormalities  of  the  hypophysis.  These  rare  pathological  condi- 
tions are  Gigantism  and  Acromegaly. 

There  are  occasional  individuals  in  whom,  either  before  or  during 
adolescence,  the  growth  of  the  skeleton  undergoes  an  extraordinary 
acceleration  so  that  they  attain  such  an  abnormal  stature  as  to  attract 
universal  attention.  Such  are  the  individuals  who  are  occasionally 
exhibited  as  "giants"  in  shows  and  fairs  (Fig.  35).  A  closer  inspection 
of  these  cases  usually  reveals  other  abnormalities  which,  in  the  adult 
at  all  events  may  be  of  two  opposite  types.  The  skin  may  be  thin, 
transparent  and  hairless,  the  extremities  small,  muscular  energy 
deficient,  the  genitals  imperfectly  developed,  and,,  according  to  Gushing, 
a  decided  intolerance  for  sugar  is  usually  also  present.  On  the  other 
hand  cases  may  be  encountered  in  which  the  reverse  of  these  charac- 
teristics may  be  noted,  the  skin  is  thick,  coarse  and  hairy,  the  extremi* 


RELATIONSHIP  OF  ENDOCRINE  ORGANS  TO  GROWTH     495 


I 


FIG.   3,5. — Preadolescent  hyperpituitarism  resulting  in  gigantism.     Height,  8  ft.  3  ii 
weight,  275  pounds.     (After  Gushing.) 


496  PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

ties  are  more  or  less  enlarged  and  the  development  of  the  sexual  organs 
may  be  exaggerated.  These  latter  symptoms  are,  however,  more 
commonly  displayed  in  the  second  type  of  anomalous  development  of 
hypophyseal  origin,  that  afforded  by  the  instances  of  Acromegaly.  In 
these  individuals  the  symptoms  do  not  usually  supervene  until  maturity 
has  been  attained  and  the  epiphyses  of  the  bones  have  hardened  so  that 
growth  in  length  is  no  longer  possible.  The  extremities  of  the  bones 
become  enlarged  so  that  the  phalanges  of  the  fingers,  for  example,  are 


FIG.  36. — Acromegalic  gigantism.     Height,    6  ft.    1  in.;  weight,   247  pounds. 
(After  Gushing.) 


spatula-shaped.  The  bodily  weight  becomes  excessive,  so  that  these 
individuals  also,  especially  if  above  the  average  in  stature,  may  from 
time  to  time  be  exhibited  as  giants.  The  features  are  coarsened  and 
thickened  and  there  is  an  extraordinary  development  of  epithelial 
tissue  and  of  epithelial  appendages.  The  development  of  hair  all  over 
the  body  may  be  so  excessive  as  to  lend  to  the  individual,  especially 
when  conjoined  with  thickened  and  distorted  features  and  massive 
development  of  the*  jaw,  a  truly  simian  appearance  (Fig.  36).  Close 


RELATIONSHIP  OF  ENDOCRINE  ORGANS  TO   GROWTH     497 


examination  usually  reveals  disturbances  of  vision  resulting  in  con- 
traction of  the  visual  field.  The  sugar  tolerance  may  be  abnormally 
high  or  abnormally  low.  Violent  headaches  and  periods  of  uncon- 
sciousness or  mental  confusion  are  frequently  experienced. 

The  ultimate  fate  of  these  cases  is  usually  heralded  by  loss  of  muscular 
power  and  a  train  of  symptoms  which  invite  the  supposition  that  the 
fundamental  condition  from  which  the  original  abnormalities  arose 


tM  • 


FIG.    37. — Dystrophia-adiposo-genitalis.     Age,    fifteen   years;    gain   of    124   pounds   in 
fourteen  months.     (After  Gushing.) 

has  become  reversed.  Examination  of  the  skull  by  means  of  the 
a>ray  usually  results,  both  in  these  cases  and  in  the  instances  of 
gigantism,  in  the  discovery  of  a  decided  enlargement  of  the  sella 
turcica,  or  bony  cavity  in  which  the  pituitary  body  is  enclosed.  Post- 
mortem examination  usually  reveals  a  tumor  in  the  neighborhood  of 
the  pituitary  body,  either  a  sarcoma  of  the  gland  itself  or  a  tumor 
exterior  to  the  gland  but  pressing  upon  it. 

It  was  pointed  out  by  Marie  that  when  the  hypophyseal  disturbance 
32 


498    PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

begins  before  adolescence  the  effect  is  to  produce  gigantism,  while  if  the 
disturbance  supervenes  after  the  attainment  of  maturity  acromegaly 
is  the  result.  He  regarded  the  two  conditions  as  differing  aspects 
of  one  and  the  same  disease,  of  which  the  symptoms  were  in  both 
instances  attributable  to  hyperactivity  of  the  hypophysis  followed 
ultimately  by  its  destruction.  Subsequent  investigators,  and  especially 
Gushing,  have  confirmed  this  view  by  more  extended  observation,  but 
they  have  also  added  a  third  type  of  pituitary  disturbance,  which  is 
designated  Frohlich's  disease,  or  Dyspituitarism.  These  cases  may  occur 
in  childhood  (Fig.  37)  or  in  adults.  They  are  characterised  by  exten- 


FIG.  38.— Fat  undersized  animal  on  left  has  undergone  partial  hypophysectomy.     Animal 
on  the  right  is  a  normal  animal  of  same  sex  and  litter.     (After  Gushing.) 

sive  deposits  of  subcutaneous  fat,  the  skin  is  thin,  transparent,  and 
hairless,  and  the  sexual  organs  and  functions  are  usually  undeveloped. 
Muscular  energy  is  at  a  very  low  level,  the  intelligence  is  usually  normal 
but  slow  These  cases  have  in  some  instances  been  markedly  alleviated 
by  the  administration  of  pituitary  tissue  or  of  pituitary  and  thyroid 
tissue  or  extracts  combined,  and  they  apparently  arise  from  deficient 

tivity  of  the  hypophysis  without  the  preliminary  stimulation  which  is 
esponsible  tor  the  characteristic  symptoms  of  gigantism  or  acromegal  v. 

Experiments  upon  animals  have  shown  us  that  while  in  mammals 
excision  of  the  posterior  lobe  or  pars  nervpsa  of  the  pituitary  body  mav 
be  endured,  complete  excision  of  both  lobes  of  the  gland  is  fa.taJ, 


RELATIONSHIP  OF  ENDOCRINE  ORGANS  TO   GROWTH     499 

tial  excision  leads  to  underdevelopment  and  particularly  to  retarded 
development  of  the  bones  (Fig.  38).  In  amphibians  complete  removal 
of  both  parts  of  the  hypophysis  is  possible  at  a  very  early  stage  of 
development  and  Smith  has  shown  that  in  hypophysectomized  tad- 
poles development  and  Metamorphosis  are  very  strikingly  retarded  in 
comparison  with  the  normals,  while  the  skin  remains  unpigmented  and 
the  tadpoles  have  the  appearance  of  albinos.  The  albinism,  but  not 
the  defective  development,  may  be  cured  or  prevented  by  the  adminis- 
tration of  posterior-lobe  extract. 

Feeding  experiments  in  which  pituitary  tissue  is  administered  to 
normal  animals  have  yielded  uniform,  but  by  no  means  striking 
results.  The  Posterior-lobe  tissue  leads  to  loss  of  weight  and  intestinal 
disturbances  which  are  not  attributable  to  or  indicative  of  any  effect 
upon  growth.  The  administration  of  Anterior-lobe  tissue  to  rats  has 
been  observed  by  Aldrich  and  by  Schafer  to  cause  retardation  of  early 
growth,  followed,  in  Schafer 's  experiments,  by  a  secondary  accelera- 
tion. Wulzen  and  Maxwell,  working  with  fowls,  likewise  obtained  retar- 

r  CRAMS, 

Normal 


IVeeKs 


4  10  ZO  30  40  £0  60 

FIG.  39. — Comparison  of  the  growth-curves  of  normal  and  of  pituitary-fed  female  white 

mice. 

dation  followed  by  acceleration  and  the  same  effect  has  been  observed 
in  mice  (Fig.  39).  The  uniform  testimony  afforded  by  all  of  these 
experiments  is  therefore  that  the  administration  of  anterior-lobe  tissue 
causes  initial  retardation  and  a  secondary  acceleration  of  growth,  but 
both  of  these  effects  are  slight. 

The  inconspicuous  character  of  these  results  is  probably  to  be 
attributed  to  the  fact  that  of  all  the  tissues  of  the  body,  the  Anterior 
Lobe  of  the  pituitary  gland  is  the  one  most  richly  supplied  with  blood. 
The  circulation  is  in  fact  extraordinarily  efficient  and  we  may  infer 
that  the  active  product  or  products  of  the  gland  leave  it  very  rapidly 
and  do  not  accumulate  therein.  Hence  the  dosage  of  the  active  mate- 
rial which  happens  to  be  present  in  the  gland  at  the  moment  of  death  of 
an  animal  may  represent  but  a  fraction  of  the  quantity  which  is  manu- 
factured and  discharged  in  the  course*  of  a  day.  When  we  administer 
pituitary  tissue  we  are  seeking  to  imitate  or  accentuate  by  a  single 
daily  administration  of  merely  residual  material,  the  action  of  a  gland 
which  is  engaged  every  moment  of  the  day  in  manufacturing  and 
discharging  the  substance  which  influences  the  growth  of  tissues; 
we  cannot,  therefore,  look  for  large  results.  As  we  shall  see,  much 
more  decisive  effects  can  be  elicited  by  the  administration  of  a  con- 


500     PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

centrated  extract  of  the  tissue,  representing  a  much  larger  dose  of  the 
fresh  tissue  than  would  be  practicable  to  employ.  The  Posterior  Lobe 
of  the  pituitary  is  but  poorly  supplied  with  bloodvessels  and  hence  the 
active  material  which  it  elaborates  accumulates  in  the  tissue  and  very 
minute  doses  of  posterior-lobe  tissue  or  extract  are  capable  of  eliciting 
the  characteristic  effects  of  Pituitrin  upon  smooth  muscular  tissue. 

The  Pineal  Gland  is  stated  by  McCord  to  have  a  decisive  influence 
upon  the  growth  of  the  Secondary  Sexual  Characters.  Tumors  of  the 
pineal  gland  have  not  infrequently  been  described,  and  are  usually 
associated  in  children  with  extraordinary  precocity  of  sexual  develop- 
ment. Either,  therefore,  the  pineal  gland  elaborates  a  principle  which 
directly  and  specifically  accelerates  the  growth  of  the  secondary  sexual 
characters,  or  else  it  operates  indirectly,  by  stimulating  the  interstitial 
cells  of  the  ovary  or  testes. 

The  relationship  of  the  Nervous  Tissues  to  the  growth  of  the  whole 
organism  is  one  which  can  by  no  means  be  overlooked  in  this  connec- 
tion. It  is,  indeed,  not  at  all  improbable  that  the  nervous  system 
performs  the  dual  role  of  a  conducting  and  coordinating  mechanism  and 
a  factory  of  endogenous  catalyzers  of  growth.  As  we  shall  see,  the 
growth-catalyzers  of  which  we  have  positive  knowledge,  Cholesterol, 
Lecithin  and  Tethelin,  are  all  lipoidal  in  character  and  these  substances, 
or  substances  related  to  them,  are  exceedingly  abundant  in  nervous 
tissues.  We  cannot  suppose  that  the  substances  which  contribute  to 
the  building  up  of  nervous  tissues  or  result  from  their  degeneration  are 
not  abundant  in  the  circulating  fluids  in  proportion  to  the  development 
of  the  nervous  tissues  or  the  ratio  of  their  mass  to  that  of  the  whole 
body,  and  several  of  them  we  know  to  exert,  and  others  we  may 
reasonably  suspect  of  exerting,  effects  analogous  to  catalysis  upon  the 
growth  of  other  tissues.  The  development  of  the  nervous  system  may 
thus  be  instrumental  in  determining  the  development  of  the  whole  body. 

THE  METABOLIC  RATE  AND  THE  PARTITION  OF  NUTRIENTS. 

The  loss  of  weight  which  occurs  in  Starvation  is  by  no  means  uni- 
formly distributed  throughout  the  body.  The  following  table  displays 
the  loss  of  substance,  in  percentages  of  the  normal  weight,  of  the 
various  tissues  of  cats  after  death  from  inanition : 

Loss  of  weight, 
Tissue  or  organ.  per  cent. 

Fat 97 

Spleen .67 

Liver .54 

Testes :..;...  40 

Muscles 31 

Kidneys 26 

Skin .  ' 21 

Intestine 18 

Lungs .      !      .      .  18 

Pancreas     ••..,%..:.....  17 

Bones    ........  14 

Heart     .      ...      .   .'.            ...........  3 

Central  nervous  system   . 


METABOLIC  RATE  AND  PARTITION  OF  NUTRIENTS      501 

It  will  be  observed  that  those  organs  which  are  most  essential  to  the 
preservation  of  existence  are  those  which  suffer  least  extensively  from 
the  unbalanced  tissue-degradation  which  results  from  the  fall  of  the 
Nutrient-level  consequent  upon  deprivation  of  food.  This  must  be  due 
to  some  definite  peculiarity  of  the  metabolism  of  those  tissues  which 
so  especially  maintain  their  weight  under  these  adverse  circumstances. 
The  nature  of  this  peculiarity  may  be  inferred  from  the  fact  that  the 
speed  of  metabolism  is  exceptionally  great  in  just  those  tissues,  the 
Heart  and  Nervous  System,  which  most  successfully  resist  the  disinte- 
gration-effects of  inanition.  Thus  the  heart  is  constantly  transforming 
large  amounts  of  potential  energy  into  mechanical  work,  the  mainte- 
nance of  life  in  the  higher  Metazoa  depends  in  fact  upon  its  doing  so, 
and  yet  it  carries  within  itself  an  extraordinarily  small  reserve  of 
energy-yielding  materials.  The  Glycogen-content  of  the  muscular 
tissues  of  the  heart,  instead  of  being  exceptionally  high,  is,  as  a  matter 
of  fact,  exceptionally  low.  The  heart  must  thus  depend  for  the 
maintenance  of  its  exertions  upon  the  direct  and  constant  withdrawal 
of  nutrient  materials  from  the  circulating  fluids.  In  so  doing  it  is 
forced  to  compete  with  all  the  other  tissues  of  the  body  and  yet  it  does 
so  with  so  much  success  that  whereas  the  majority  of  the  other  tissues 
lose  a  very  considerable  part  of  their  weight,  the  heart  maintains  the 
integrity  of  its  substance  until  death  is  imminent.  This  implies  that 
the  rate  of  utilization  of  nutrients  by  the  heart  must  greatly  exceed 
that  of  the  other  tissues,  so  that  the  foodstuffs  are  appropriated  in 
advance  of  the  ability  of  other  tissues  to  do  so. 

The  high  Metabolic  Rate  of  the  central  nervous  system  may  be  inferred 
from  the  fact  that  its  consumption  of  oxygen  is  exceptionally  great. 
The  first  effect  of  deprivation  of  oxygen  is  to  arrest  the  higher  activities 
of  the  central  nervous  system  and  those  substances  which  paralyze 
the  oxidizing  enzymes,  such  as  the  Cyanides,  arrest  the  activities  of  the 
central  nervous  system  before  any  other  tissue  is  affected  to  a  com- 
parable degree.  The  intensity  of  Oxidations  in  the  central  nervous 
system  testifies  to  the  rapidity  of  the  destruction  of  its  constituents. 
The  fact  that  it  maintains  its  integrity  even  in  starvation,  therefore, 
implies  a  proportionate  rapidity  of  reconstruction. 

The  synthesis  of  the  various  tissues  of  the  body  from  the  foodstuffs 
which  are  contained  in  the  circulating  fluids  may  be  regarded  as  a 
multitude  of  parallel  reactions,  all  consuming  similar  substrates 
although  not  in  identical  amounts  and  proportions.  Now  in  any  group 
of  Parallel  Reactions,  that  is,  of  reactions  which  are  occurring  simul- 
taneously and  consuming  the  same  raw  materials,  each  substrate  which 
enters  into  the  reactions  is  shared  between  them  in  proportion  to  the 
velocity  with  which  they  occur.  The  various  reactions  proceed  at  their 
own  independent  rates  and  if  the  quantity  of  materials  available  for 
transformation  were  unlimited,  each  reaction,  or  the  synthesis  of  each 
particular  kind  and  type  of  tissue,  would  go  forward  at  the  same  speed 
as  it  would  if  the  other  tissue-syntheses  were  not  occurring  simul- 


502     PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

taiK'ously.  The  quantity  of  available  substrates  or  Nutrient -level  of 
the  tissue-fluids  is,  however,  not  unlimited  but  adjusted,  as  we  have 
seen,  by  a  dynamic  equilibrium,  to  the  average  needs  of  the  body  as  a 
whole.  In  the  competition  for  these  materials,  therefore,  the  most 
specifically  rapid  syntheses  will  have  a  decided  advantage  over  the 
specifically  slower  syntheses,  and  when  the  nutrient-level  sinks  below 
the  normal,  as  in  starvation,  the  more  rapidly  metabolizing  tissues 
will  maintain  their  integrity  for  relatively  prolonged  periods  at  the 
expense  of  the  more  slowly  metabolizing  tissues. 

If  we  now  turn  to  the  question  of  the  origin  of  the  varying  metabolic 
rate  of  different  tissues,  we  can  only  infer  that  the  rapidly  metabolizing 
tissues  produce  Endogenous  Catalyzers  of  growth  which  are  either  more 
efficient  accelerators  than  those  which  are  produced  by  other  tissues 
or  else  are  produced  in  greater  amount.  We  may  thus  clearly  look  to 
the  nervous  system  and  the  tissues  of  the  heart  as  the  origin  of  very 
powerful  or  abundant  catalyzers  of  growth.  Since  the  majority  of 
catalyzers,  and  probably  the  growth-catalyzers  also,1  accelerate  both 
the  forward  and  the  backward  reaction,  both  the  anabolism  and  the 
catabolism  of  such  tissues  are  exceptionally  rapid. 

Since  the  effect  of  starvation  is  to  favor  the  rapidly  metabolizing 
tissues  at  the  expense  of  those  of  slower  metabolic  rate  the  result  must 
be  to  increase  the  proportion  of  rapidly  metabolizing  tissues  in  an 
animal  and  the  production  of  growth-catalyzers  per  kilo  of  body-weight. 
Corresponding  with  this  fact  Osborne  and  Mendel  found  that  a  period 
of  starvation  greatly  improves  the  subsequent  utilization  of  foodstuffs, 
so  that  in  a  growing  rat  the  total  growth  attained  in  a  period  of  starva- 
tion followed  by  a  period  of  feeding  may  exceed  that  attained  by  normal 
animals  in  a  like  period  of  time.  A  second  period  of  starvation  even 
enhances  this  effect.  The  same  effect  may  often  be  noted  in  infants 
as  a  result  of  a  period  of  subnutrition  or  of  a  lowered  nutritional  level 
due  to  the  enhanced  exogenous  metabolism  in  fevers. 

From  quite  another  avenue  of  experimental  investigation  the  conclu- 
sion may  also  be  drawn  that  a  period  of  starvation  increases  the  pro- 
portion of  vigorously  metabolizing  tissues  in  the  body.  Embryonic 
Tissues  and  rapidly  growing  tissues  generally  have  been  shown  by  many 
observers,  and  particularly  by  Cramer,  to  contain  a  high  proportion  of 
Water,  while  those  which  metabolize  most  slowly  and  suffer  most  in  any 
severe  competition  for  nutrients  contain  a  relatively  low  proportion 
of  water.  The  nervous  system,  for  example,  contains  an  exceptionally 
high  percentage  of  water.  Now  Aron  has  shown  that  a  period  of  starva- 
tion or  subnutrition  leads  both  in  children  and  in  animals  to  a  greater 
loss  of  nitrogen  and  calories  than  would  normally  be  equivalent  to  the 
loss  of  body-weight;  in  other  words  the  tissues  are  becoming  progres- 
sively more  dilute  and  of  less  calorific  value. 

We  are  led  again  in  this  connection  to  recall  the  important  observa- 

1  We  may  infer  this  from  the  symmetry  of  the  curve  of  growth. 


CATALYZERS  OF  GROWTH  503 

tion  of  Child  that  starving  planarians  undergo  retrogression  to  a 
relatively  embryonic  eharaeter.  Child  accounts  for  this  Rejuvenescence 
by  the  sweeping  out  from  the  cell  of  accumulations  of  colloidal  sub- 
stances which  impede  the  cell-activities  and  are  consumed  in  starvation 
for  purposes  of  furnishing  energy.  The  nature  of  the  impediment 
constituted  by  these  substances  is,  however,  by  no  means  clear;  but 
it  may  very  conceivably  be  possible  that  a  high  proportion  of  water  is 
essential  to  the  production  of  growth-catalyzers  in  abundance.  The 
relative  rejuvenescence  of  metazoa  by  starvation  is,  however,  more 
probably  to  be  attributed  to  the  ascendency  in  mass  and  numbers 
acquired  by  the  tissues  which  are  normally  possessed  of  a  high  metabolic 
rate,  which  enables  them,  when  food  is  readmitted,  to  push  forward  all 
of  the  processes  of  growth,  including  the  growth  of  slowly  metabolizing 
and  water-poor  tissues,  with  unusual  energy. 

CATALYZERS  OF  GROWTH. 

If  a  catalyzer  is  of  the  "typical"  variety  and  is  not  in  any  degree 
consumed  during  the  reaction  which  it  accelerates,  then  it  necessarily 
follows  that  it  cannot  alter  the  final  Equilibrium  of  the  reaction,  for  a 
shift  in  chemical  equilibrium  means,  generally  speaking,  that  heat  is 
either  produced  or  absorbed  and  the  equivalent  in  work  or  heat  must 
be  supplied  by  agencies  external  to  the  reaction  itself,  or  by  spme 
other  collateral  chemical  reaction.  Since  the  catalyzer  introduces  no 
condition  not  implied  in  its  presence,  the  energy-change  involved  in 
a  shift  of  equilibrium  would  of  necessity  be  equated  by  a  change  in  the 
energy-content  of  the  catalyzer  which  could  only  be  supplied  by  its 
chemical  transformation,  i.  e.,  by  consuming  it.  It  follows,  of  course, 
that  a  catalyzer  cannot  initiate  a  chemical  reaction  which  is  not  already 
proceeding,  however  slowly,  in  its  absence. 

If  Endogenous  Catalyzers  of  growth  really  exist,  therefore,  we  should 
expect  them  to  display  the  following  characteristics,  distinguishing  them 
more  or  less  clearly  from  the  growth-substrates : 

1.  Since  these  catalyzers  are  not  the  only,  nor  necessarily  quan- 
titatively important  constituents  of  the  tissues  which  are  the  sum 
of  the  products  of  growth,  it  follows  that  the  effect  of  catalyzers  of 
growth  may  be  totally  disproportionate  to  their  nutritive  (i.  e.,  calorific) 
value. 

2.  The  ultimate  growth  attained  by  two  groups  of  animals  under  the 
influence  of  unequal  amounts  of  the  catalyzer  may  be  expected  to  tend 
toward  equality,  since  the  ultimate  station  of  equilibrium  of  a  reaction 
is  unaffected  by  a  catalyzer,  although  the  velocity  with  which  equi- 
librium is  attained  may  be  profoundly  affected.     This  tendency  is, 
however,  limited  by  three  groups  of  factors,  namely  (a)  the  mechanical 
delay  or  prevention  of  growth  which  may  be  imposed  upon  an  animal 
by  the  formation  of  a  skeleton  or  of  a  circulatory  or  respiratory  system 
of  limited  dimensions,     (6)  By  the  unequal  effect  of  catalyzers  upon 


504    PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

different  types  of  tissue,  leading,  as  we  shall  see,  to  the  favoring  of 
tissues  of  high  metabolic  rate,  other  tissues  being  retarded  in  their 
growth  by  the  successful  competition  of  the  favored  tissues,  (c)  By 
the  onset  of  senescence,  which  ultimately  terminates  and  prevents  the 
full  fruition  of  the  growth-process. 

3.  Growth  may  take  place  in  the  absence  of  catalyzers  added  to  the 
diet,  since  they  are  produced  by  the  growing  tissues  themselves  or  by 
organs  to  which  this  particular  function  has  wholly  or  partially  been 
delegated.    The  growth-catalyzers  are  therefore  not  essential  dietary 
constituents  in  the  sense  in  which  the  growth-substrates  are  essential. 

4.  Growth-catalyzers  may  be  expected  to  appreciably  influence  the 
rate  of  growth  even  when  superadded  to  an  already  varied  and  abun- 
dant diet,  whereas,  in  normal  animals,  -provided  all  of  the  growth- 
substrates  be  present  in  the  dietary  in  abundance  the  addition  of  a 
particular  substrate  in  excess  merely  leads  to  enhanced  exogenous 
metabolism  of  that  foodstuff  and  not  to  enhanced  utilization  for  tissue- 
building. 

5.  There  is  no  reason  to  assume  that  the  growth-catalyzer  for  any 
one  group  of  tissues,  is  necessarily  identical  with  that  for  any  other. 
On  the  contrary  we  have  evidence,  as  in  the  effect  of  the  interstitial 
cells  of  the  testes  or  ovaries  upon  the  growth  of  secondary  sexual  charac- 
ters, and  of  the  secretions  of  the  corpora  lutea  upon  the  development 
of  the  placenta,  that  growth-catalyzers  may  exist  which  are  specific 
for  individual  tissues.     Growth-substrates,  on  the  contrary,  facilitate 
growth  as  a  whole,  and  although  at  a  low  nutrient-level  the  high 
metabolic  rate  of  certain  tissues  may  enable  them  to  appropriate  the 
lion's  share  of  the  foodstuffs,  yet  under  normal  conditions  all  tissues 
are  similarly  affected  in  differing  degrees  by  the  various  growth- 
substrates. 

6.  Growth-catalyzers  will  be  unable  to  initiate  new  growths,  just 
as  other  catalyzers  are  unable  to  initiate  the  reactions  which  they 
accelerate. 

Several  substances  have  been  discovered  to  influence  the  rate  of 
growth  of  animals  and  of  individual  tissues  when  administered  in 
dosages  which  are  devoid  of  nutritive  significance  and  which  correspond 
in  all  of  the  particulars  enumerated  above  with  the  anticipated  proper- 
ties of  growth-catalyzers.    Thus  if  Cholesterol  be  administered  either 
by  mouth  or  subcutaneously  to  animals  which  have  been  previously 
inoculated  with  pieces  of  Carcinoma-tissue,  the  growth  of  the  tumor 
.s  enormously  accelerated  and  out  of  all  proportion  to  the  nutritive 
value  which  the  minute  dosage  of  cholesterol  which  is  requisite  might 
be  supposed  to  have,  if  we  did  not  know  that  as  a  matter  of  fact  the 
greater  proportion  of  administered  cholesterol  is  excreted  unchanged. 
Not  only  is  the  rate  of  growth,  of  the  primary  tumor,  as  estimated  by 
s  increase  of  diameter,  increased  by  one  or  two  hundred  per  cent., 
the  growth  of  Metastases  or  offshoots  of  the  tunior  in  distant 
organs  and  the  percentage  of  animals  displaying  metastases  are  very 


CATALYZERS  OF  GROWTH 


505 


remarkably  increased.  Sweet,  Corson-White  and  Saxon  had  a  strain 
of  carcinoma  which  had  never  been  known  in  their  experience  to  yield 
metastases  in  rats.  They  administered  cholesterol  by  mouth  to  a  large 
number  of  rats  inoculated  with  this  tumor  and  obtained  metastases 
in  over  ninety  per  cent,  of  the  animals.  It  has  also  been  shown  by 
Browder  that  cholesterol  has  a  remarkable  influence  upon  the  rate  of 
multiplication  of  the  infusorian  Paramecium,  increasing  the  number 
of  generations  produced  in  a  given  period  by  several  hundred  per  cent. 
If  cholesterol  be  administered  to  young  mice  in  dosages  of  40  mgm. 
per  day,  however,  a  result  is  obtained  which  is  at  first  sight  rather 
surprising,  for  the  growth  of  the  animals,  instead  of  being  accelerated, 
is  very  markedly  retarded  during  the  early  weeks  of  the  third  growth- 
cycle  (fifth  to  fifteenth  week)  and  subsequently  undergoes  a  secondary 
acceleration  which,  however,  never  makes  up  for  the  ground  lost  during 


d 


70    80     90    100    110    120    130    140    160 

WEEKS 


5  10  15  20  25  30     40     50 


FIG.  40. — Influence  of  cholesterol  upon  the  growth  of  male  white  mice.     Dosage, 
40  mgms.  per  day.     The  vertical  cross-mark  indicates  average  duration  of  life. 

the  period  of  initial  retardation  (Fig.  40).  Now  when  cholesterol  is 
administered  in  unusual  amounts  to  animals  the  excretory  mechanisms 
prove  insufficient  and  large  deposits  are  formed  in  a  variety  of  organs, 
particularly  the  liver,  spleen  and  suprarenal  capsules,  and  it  might  be 
imagined  that  this  or  some  other  deleterious  effect  of  cholesterol, 
superadded  to  its  effect  upon  growth  is  responsible  for  the  retardation 
of  the  growth  in  weight  of  animals  to  which  its  administration  leads. 
This,  however,  is  not  the  case,  for  this  effect  of  cholesterol  is  merely 
a  particular  instance  of  the  general  action  of  growth-catalyzers  upon 
the  adolescent  growth  of  animals. 

It  will  be  recollected  that  the  administration  of  the  tissue  of  the 
Anterior  Lobe  of  the  Pituitary  Body  to  growing  animals  produces  a  like 
unexpected  result,  namely  a  retardation  of  the  early  adolescent  growth 
followed  by  a  secondary  acceleration.  Now  hypophyseal  tissue,  when 


.")()()     1>R()CESSE8  INFERRED  FROM  INDIRECT  OBSERVATION 

emulsified  and  administered  by  hypodermic  injection,  brings  about  an 
acceleration  of  the  growth  of  inoculated  carcinoma  in  rats  which  is 
just  as  marked  as  that  which  is  caused  by  cholesterol.  By  extraction 
with  alcohol  and  subsequent  precipitation  with  ether  a  substance  is 
obtained  from  the  dried  tissue  of  the  anterior  lobe  of  the  pituitary 
body  which  has  been  designated  Tethelin.  This  substance  is  evidently 
a  lipoid,  for  it  yields  fatty  acids  on  hydrolysis,  but  it  is  a  lipoid  of  very 
exceptional  physical  and  chemical  characteristics.  It  is  soluble  in  water, 
alcohol  or  ether,  but  insoluble  in  a  mixture  of  certain  definite  pro- 
portions of  alcohol  and  ether.  It  is  present  in  ox-glands  to  the  extent 
of  about  0.7  per  cent,  of  the  fresh  anterior-lobe  tissue.  The  adminis- 
tration of  four  milligrams  of  this  substance  per  day  to  mice  from  five 
weeks  of  age  onward  produces  a  most  decisive  change  in  the  velocity 
and  time-relations  of  growth.  The  effect  is  similar  in  kind  to  that  of 


d 


70  80 

WEEKS 


FIG.  41. — Influence  of  tethelin  upon  the  growth  of  male  white  mice.     The  vertical 
cross-mark  indicates  average  duration  of  life. 

the  administration  of  pituitary  tissue  itself,  that  is,  initial  retardation 
followed  by  acceleration,  but  both  effects  are  exaggerated  so  greatly  as 
to  involve  total  distortion  of  the  curve  of  growth,  the  second  growth- 
cycle  appearing  to  be  prolonged  while  the  third  or  adolescent  cycle  is 
abbreviated  and  accelerated*  (Fig.  41).  The  quantitative  difference 
between  the  growth-effects  obtained  with  tethelin  and  observed  in 
anterior-lobe  tissue  administration  are  attributable  to  the  difference 
m  the  dosage  of  tethelin  which  is  received  in  the  two  cases.  It  is  not 
practicable,  for  example,  to  administer  much  more  than  a  twelfth  of 
a  fresh  ox-gland  per  day  to  mice,  because  the  quantity  of  meat  consumed 
would  otherwise  constitute  an  important  abnormality  in  the  diet. 
Phis  amount  of  pituitary  tissue,  however,  contains  only  between  eight 
and  nine-tenths  of  a  milligram  of  tethelin,  or  one-fifth  the  amount 
administered  in  the  experiments  cited  above. 


CATALYZERS  OF  GROWTH 


507 


The  influence  of  tethelin  upon  the  growth  of  mice  is  therefore 
similar  to  the  effect  of  administering  cholesterol,  save  that  results  are 
attained  by  administration  of  tethelin  with  a  tenth  of  the  dosage  that 
would  be  requisite  in  the  case  of  cholesterol.  It  is  very  significant, 
therefore,  that  the  action  of  tethelin  upon  inoculated  Carcinoma  in 
rats  again  reproduces  the  effects  of  cholesterol  (Fig.  42.) 

Even  more  striking  than  its  effect  upon  the  growth  in  weight  of  the 
animals  is,  however,  the  effect  of  tethelin  upon  the  general  contour 
and  appearance  of  mice  to  which  it  has  been  administered  continuously. 
The  tethelin-fed  animals  are  remarkably  robust  and  compact  in  build. 
Weight  for  weight  they  are  smaller  and  size  for  size  much  heavier  than 
normal  animals.  The  contours  of  their  surface  are  more  rounded  and 
fully  adult  animals  retain  a  youthful  appearance  which  is  soon  lost  in 


80- 


60- 


4-0 


20- 


Per  cent  Increase  over 
Diameter   at    21  days 


Days  after  inoculation 


21 


23 


25 


28 


30 


FIG.  42. — The  acceleration  of  the  growth  of  carcinomata  (in  rats)  by  hypodermic 
administrations  of  tethelin. 

normal  animals.  The  coats  of  the  males,  even  at  fourteen,  months  of 
age,  retain  the  glossy,  silky  appearance  of  the  coats  of  young  animals 
or  of  females,  while  six  months  or  more  prior  to  this  age  the  coats 
of  normal  males  are  already  shaggy,  staring,  and  discolored.  These 
differences  are  clearly  displayed  in  the  accompanying  photograph, 
in  which  a  normal  and  a  tethelin-fed  male  of  the  same  age  (one  year) 
and  of  the  same  weight  (28.0  gm.)  are  compared  (Fig.  43).  The 
normal  animal  on  the  left  has  a  shaggy,  staring  and  discolored  coat, 
while  the  tethelin-fed  animal  has  a  smooth,  glossy  and  pure  white  coat. 
The  normal  animal  is  irregular  in  outline  and  loosely  built,  while  the 
contour  of  the  tethelin-fed  animal  is  rounded  and  its  build  is  compact. 
In  each  of  these  three  instances  of  growth-catalysis,  therefore,  we 
meet  with  the  apparently  contradictory  fact  that  while  the  growth  of  a 


508     PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

neoplasm  (carcinoma)  is  accelerated  by  the  catalyzer,  the  growth  of 
young  animals  prior  to  sexual  maturity  is  retarded.  It  might  be 
imagined  that  this  constituted  evidence  of  a  fundamental  difference 
between  the  metabolism  of  malignant  tissue  and  that  of  normal  tissue. 
This  inference  would  not  be  justified,  however,  because  in  the  first 
place  no  other  evidence  of  a  fundamental  difference  between  the  growth 
of  malignant  and  of  normal  tissues  has  ever  been  advanced  and,  in  the 
second  place,  the  accelerative  action  of  these  catalyzers  upon  growth 
is  not  by  any  means  confined  to  the  growth  of  malignant  tissues. 
Thus  cholesterol  accelerates,  as  we  have  seen,  the  division-rate  in 


FIG.  43. — Comparison  of  a  normal  (left)  and  a  tethelin-fed  (right)  male  white  mouse, 
both  one  year  old  and  28  grams  in  weight.  Note  the  smooth  coat  and  compact  form  of 
the  tethelin-fed  mouse  as  contrasted  with  the  loose  form  and  rough  coat  of  the  normal 
animal. 

Paramecia.  Our  clinical  experience  abundantly  confirms  the  fact  that 
hyperactivity  of  the  pituitary  body  leads  to  abnormally  rapid  develop- 
ment of  bony  and  Epithelial  Tissues  and,  finally,  tethelin  markedly 
accelerates  the  regeneration  of  epithelium  lost  by  injury  and  the  regain 
of  weight  lost  during  a  period  of  inanition  after  the  readmission  of  food. 
The  action  of  tethelin  in  hastening  the  repair  of  epithelial  lesions  is  so 
decided  that  it  has  been  proposed  as  a  means  of  accelerating  the  repair 
of  slowly-healing  wounds,  such  as  the  leg-ulcers  which  may  result  from 
varicose  veins. 

We  have  the  apparently  opposed  facts,  therefore,  that  cholesterol 
and  tethelin  definitely  accelerate  the  growth  of  certain  types  of  tissue, 


CATALYZERS  OF  GROWTH  509 

while  the  growth  of  the  entire  animal  is  retarded.  Evidently,  there- 
fore, there  are  in  the  body  certain  other  and  relatively  bulky  tissues  of 
which  the  growth  is  directly  or  indirectly  retarded  by  tethelin. 

The  most  probable  reason  for  this  retardation  lies  in  the  varying 
Metabolic  Rates  of  the  different  tissues  of  the  body  and  their  consequent 
differing  success  in  the  competition  for  nutrients.  There  are,  broadly 
speaking,  two  easily  distinguishable  groups  of  tissues  in  the  animal 
body  which  differ  fundamentally  in  function  and  metabolism.  These 
are  on  the  one  hand  the  Parenchymatous  Tissues,  which  are  essentially 
cellular,  self-maintaining  cells  derived  from  the  ectoderm  and  entoderm 
of  the  three  embryonic  layers  and  on  the  other  hand  a  variety  of  tissues 
which  originate  mainly  but  not  exclusively  from  the  mesoderm  and 
constitute  the  Sclerenchyma  or  tissues  of  primarily  structural  or  archi- 
tectural significance.  These  latter  tissues  are  dependent.  They  can 
only  arise  through  the  activities  of  nucleated  living  cells,  of  which  they 
constitute  outgrowths,  secretions,  or  products  of  retrogressive  change. 
Of  this  character,  for  example,  are  the  various  fibrous  tissues,  the 
elastic  and  calcified  tissues,  and  the  ligaments,  tendons  and  other 
structures  which  bind  together  and  support  the  tissues  of  more  varied 
and  complex  function.  The  sclerous  tissues  have  a  low  Metabolic  Rate, 
are  among  those  which  lose  most  heavily  in  the  competition  for  a  sub- 
normal supply  of  nutrients  and,  since  they  are  as  a  rule  devoid  of  the 
powrer  of  multiplication  or  even  of  repair  without  the  intervention  and 
assistance  of  other  cells,  we  may  legitimately  infer  that  they  do  not 
produce,  as  the  parenchymatous  tissues  do,  Endogenous  Catalyzers 
which  accelerate  their  synthesis  and  degradation.  In  fact  since  their 
synthesis  is  accomplished  by  other  cells  there  would  be  no  particular 
purpose  served  by  their  doing  so.  Thus  the  horny  cells  of  superficial 
epidermis,  which  have  lost  the  power  of  reproduction  and  growth  in  the 
course  of  the  degenerative  changes  which  have  resulted  in  their  trans- 
formation into  Keratin,  are  renewed  from  time  to  time  by  the  multi- 
plication of  the  cells  of  the  Malpighian  layer  of  the  deeper  epidermis. 
Cartilage  and  bone  are  similarly  formed  from  cellular  tissues  and  the 
fibrous  tissues  are  excretions  or  transformation-products  of  the  Fibro- 
blasts  from  which  they  originate.  Even  the  muscular  tissues  may  in 
like  manner  originate  from  special  cells  which  have  retained  the 
potentiality  of  reproduction.  But  if  these  tissues  do  not  produce 
endogenous  catalyzers  and  in  many  cases  cannot  form  the  material  of 
which  they  are  composed,  it  is  evident  that  growth-catalyzers  from 
other  sources  can  only  affect  their  development  in  the  indirect  fashion 
of  promoting  the  growth  or  multiplication  of  the  cells  or  other  tissues 
from  which  they  arise. 

A  catalyzer  of  growth  may  accelerate  the  formation  of  parenchyma- 
tous tissues,  but  its  exceptional  abundance  or  potency  may  actually 
retard  the  growth  of  the  tissues  which  are  not  directly  affected  by  it, 
through  the  deflection  of  nutrients  to  the  parenchymatous  elements. 
An  important  proportion  of  the  total  increment  in  weight  of  an  animal 


510     PROCESSES  INFERRED  FROM  INDIRECT   OBSERVATION 

during  the  adolescent  growth-cycle  is  the  formation  of  Connective 
Tissues1  and  if  the  development  of  certain  of  these  be  retarded  in  the 
manner  indicated,  it  may  readily  be  understood  how  the  rate  of  growth 
of  the  animal  as  a  whole,  estimated  by  its  weight,  is  retarded  although 
the  growth  of  its  parenchymatous  tissues  may  be  considerably  acceler- 
ated. That  this  is  probably  the  correct  interpretation  of  the  facts  is 
furthermore  shown  by  the  effect  of  discontinuing  the  administration  of 
tethelin  to  mice  after  the  initial  retardation  of  growth  has  become  well 
marked.  The  secondary  acceleration  of  growth  which  succeeds  the 
retardation  in  animals  which  received  tethelin,  cholesterol  or  pituitary 
tissue  throughout  their  lives  is,  in  this  event  very  much  enhanced,  so 
that  the  effect  of  the  initial  retardation  of  growth  is  not  only  fully 


FIG.  44. — Showing  the  effect  of  a  brief  period  (five  weeks)  of  administration  of  tethe- 
lin upon  the  subsequent  growth  of  mice.  Animal  on  the  left  (31  grams)  is  the  average 
tethelin-treated  animal  at  five  hundred  days.  On  the  right  (25  grams)  an  average 
normal  animal  of  same  age. 

compensated,  but  a  supernormal  accretion  of  weight  occurs,  carrying 

the  animals  far  beyond  the  average  of  normal  animals  of  the  same  age. 

This  is  strikingly  shown  in  the  preceding  photograph  (Fig.  44),  in  which 

a  female  mouse  of  average  normal  weight  at  five  hundred  days  of  age 

=  25  grams)  is  compared  with  a  female  representing  the  average  weight 

=  33  grams)  of  animals  which  had  received  four  milligrams  of  Tethelin 

daily  from  the  fifth  to  the  thirteenth  week  of  age;  the  administrations 

being  then  discontinued.     The  remarkable  overgrowth  which  is  thus" 

attained  is  evident  even  in  the  average  animal  displayed  in  the  photo- 

1  Tims  BischolT  (Volt's  Handbuch  der  Physiologic,  Bd.  0,  p.  .511)  finds  that  the 
muscular,  skeletal  and  fatty  tissues  comprise  76  per  cent,  of  the  weight  of  the  adult 

W       AttJt8'  Cent'.°f  the  W6ight  °f  the  newborn-     Rubner  estimates  that  a  man 
08  contains  37.8  kilos  of  cell  mass  of  which  40  per  cent,  is  muscular  tissue 


CATALYZERS  OF  GROWTH  511 

graph,  but  one-eighth  of  the  animals  so  treated  actually  attained  weights 
in  excess  of  forty  grams,  a  weight  which,  it  may  be  stated,  no  normal 
female  mouse  ever  attains.  This  remarkable  overgrowth  is  probably 
attributable  to  the  preceding  development  of  parenchymatous  tissues. 
The  removal  of  the  stimulus  which  enabled  them  to  predominate  in 
the  struggle  for  nutrients  gives  the  sclerous  tissues  the  opportunity 
to  develop,  and  the  reattainment  of  normal  proportionality  between 
the  sclerenchyma  and  parenchyma  finally  enables  the  stimulation  of 
growth  which  has  actually  occurred  to  find  expression  in  the  super- 
normal weight  of  the  animal  as  a  whole.  The  occurrence  of  Acromegaly 
in  man  may  actually  indicate  therefore,  not  a  present  hyperactivity  of 
the  hypophysis,  but  a  preceding  hyperactivity,  succeeded,  before  the 
onset  of  the  acromegalic  symptoms,  by  a  normal  or  even  subnormal 
activity  of  the  gland. 

It  is  a  noteworthy  fact  that  although  the  administration  of  Choles- 
terol or  Tethelin  to  normal  animals  which  have  been  inoculated  with 
Carcinoma  leads  to  acceleration  of  the  growth  of  the  neoplasm,  yet  it 
has  so  far  proved  impossible,  despite  many  trials,  to  induce  the  spon- 
taneous development  of  tumors  in  animals  by  the  administration  of 
these  substances.  The  percentage  of  mice  which  develop  carcinoma 
is  the  same  in  animals  which  have  received  cholesterol  or  tethelin  for 
the  greater  part  of  their  lives  as  it  is  in  normal  animals.  In  other 
words  these  substances,  like  the  catalyzers  with  which  we  are  familiar 
in  other  chemical  transformations,  are  unable  to  initiate  the  reaction 
which  they  accelerate.1  Moreover  the  spontaneous  development  of 
carcinoma  is  even  greatly  delayed  and  the  growth  of  the  neoplasm 
when  it  has  arisen  is  very  much  slowed  by  the  continuous  administra- 
tion of  tethelin  to  animals.  It  would  appear  that  the  continuous 
administration  of  tethelin  results  in  such  a  disproportionate  develop- 
ment of  parenchymatous  tissues  that  they  are  enabled  to  compete 
successfully  with  the  neoplasm  for  the  nutrients  in  the  tissue-fluids, 
whereas  in  the  normal  animal  the  neoplasm  shares  with  the  limited 
proportion  of  parenchyma  the  advantages  of  enhanced  catalysis  of  the 
growth-processes. 

Carcinoma  is  essentially  a  disease  of  old  age  and  the  investigations 
of  Wacker  have  shown  that  the  cholesterol-content  of  the  subcutaneous 
fats  is  exceptionally  high  in  elderly  people  and  in  persons  afflicted  with 
carcinoma.  Luden  has  also  found  that  cholesterol  is  exceptionally 
abundant  in  the  blood  of  individuals  suffering  from  carcinoma,  wrhile 
the  oxidation-products  of  cholesterol  which  yield  Lifschiitz's  reac- 
tion without  preliminary  treatment  with  oxidizing-agents,  which  are 
abundant  in  normal  blood,  are  absent  or  scanty  in  the  blood  of  carci- 
nomatous  individuals. 

1  Erdmarm  has  described  an  innoculable  tumor  which  was  produced  by  the  inocu- 
lation of  foreign  non-malignant  tissue  followed  by  an  induced  inflammatory  reaction 
;  u  H!  administration  of  tethelin,  but.  tethelin  ajqne  was  ineffective. 


512    PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

OLD  AGE  AND  SENESCENCE. 

The  leading  characteristic  of  old  age  is  the  low  average  Metabolic 
Rate  of  the  tissues.  From  maturity  to  old  age  the  calorific  output 
steadily  diminishes,  the  total  reduction,  according  to  Du  Bois,  being 
about  thirteen  per  cent,  by  eighty  years  of  age  in  men.  This  dimin- 
ished metabolism,  if  it  is  not  accompanied  by  a  corresponding  diminu- 
tion of  intake,  may  lead  to  the  formation  of  extensive  deposits  of  fat 
and  the  Obesity  which  occurs  in  a  certain  percentage  of  elderly  indi- 
viduals. In  general,  however,  the  decreased  metabolic  rate  is  accom- 
panied by  a  progressive  loss  of  body-weight.  In  man  the  senescent 
loss  of  body-weight  begins  relatively  early,  but  proceeds  very  slowly, 
so  that  it  only  becomes  notable  at  an  age  in  excess  of  the  mean  duration 


9 


1C 
in 


5  10  15  20  25  30    40     50    60     70    80     00    100    110    120    130    140    150 

WEEKS 

FIG.  45. — Growth  curve  of  normal  female  white  mice  from  four  weeks  until  death 
of  the  last  surviving  animal.  The  vertical  cross-mark  indicates  average  duration  of 
life. 

of  life.  In  the  mouse,  on  the  contrary,  the  Senescent  Loss  of  Weight 
is  relatively  sudden  and  rapid  and  is  quite  marked  before  the  mean 
duration  of  life  is  attained.  This  is  illustrated  by  the  accompanying 
curve  (Fig.  45),  which  displays  the  growth  and  senescence  of  female 
white  mice  from  four  weeks  until  the  termination  of  the  observations  by 
the  death  of  the  last  surviving  animals.  Deaths  from  epidemic  infec- 
tion were  excluded  by  the  technique  of  the  experiments.  The  terminal 
fluctuations  of  the  curve  are  due  to  the  irregularly  occurring  deaths  of 
animals  in  which  the  process  of  senescence  has  been  most  rapid  and 
which  have  lost  most  weight.  The  survivors  therefore  represent  an 
earlier  or  less  complete  stage  of  senescence  than  those  which  have  died, 
and  each  group  of  late  deaths  is  consequently  accompanied  by  a  rise 
in  the  weight-curve  of  the  survivors.  Each  rise,  however,  is  succeeded 
by  a  fall,  which  is  even  more  rapid  than  the  preceding  one,  indicating 


OLD  AGE  AND  SENESCENCE  513 

that  the  process  of  senescence  is  in  reality  continuous,  and,  moreover, 
that  it  proceeds  with  a  regularly  increasing  velocity  which  depends 
upon  the  age  rather  than  upon  the  weight  of  the  animals.  The  same 
characteristics  are  displayed  by  the  curves  in  Fig.  41  on  p.  506. 

There  is  no  particular  reason,  implied  in  the  nature  of  an  auto- 
catalytic  process,  why  the  mass  of  its  product  should  diminish.  In 
fact,  the  station  of  Equilibrium  in  a  purely  autocatalytic  process,  un- 
complicated by  side  reaction,  is  asymptotically  approached  and  never 
actually  attained,  so  that  the  total  mass  of  product,  so  far  from  decreas- 
ing at  the  apparent  close  of  the  reactions,  is  actuallly  increasing  at 
an  infinitesimal  rate.  The  process  of  growth,  however,  although  it  is 
autocatalyzed,  does  not  conform  to  this  particular  characteristic  of 
autocatalytic  reactions  and,  a  maximum  yield  of  product  having  been 
attained,  the  tissues  slowly  disintegrate,  even  gathering  speed  as  time 
proceeds,  until,  if  no  other  factor  intrudes  to  terminate  life,  Senile  Atrophy 
of  the  tissues  leads  to  irreparable  weakening  of  some  essential  organ. 

A  variety  of  hypotheses  have  been  advanced  to  account  for  the 
phenomena  of  senescence  which,  even  if  all  other  dangers  of  life  could 
be  surmounted,  would  set  an  inevitable  term  to  existence.  A  very 
natural  supposition  is  that  proposed  by  Biitschli,  that  death  is  due  to 
the  exhaustion  of  a  certain  substance — the  "life  ferment" — which  is 
gradually  used  up  during  life.  We  cannot  disassociate  senescent 
atrophy  from  senescent  death,  however,  since  the  death  of  aged  indi- 
viduals is  obviously  determined  by  the  progressive  atrophy  or  degener- 
ation of  essential  tissues.  Now  senescent  atrophy  is  attributable  to  the 
inability  of  the  tissues  to  maintain  their  weight  and  we  must  therefore, 
in  the  terms  of  Biitschli's  hypothesis,  suppose  that  the  gradual  con- 
sumption of  an  essential  substance  which  was  originally  contained  in 
the  germ-cells  and  can  be  manufactured  only  by  them,  has  deprived 
the  tissues  of  the  power  to  form  new  protoplasm.  Now  this  is  not  the 
case,  for  even  in  old  age,  injury,  or  removal  of  the  Products  of  Growth, 
will  institute  vigorous  Regeneration  and  repair.  The  capacity  to  grow 
is  not  lost  or  even  impaired  by  age.  Thus  Osborne  and  Mendel  have 
maintained  rats  in  an  infantile  stage  of  development  by  depriving  them 
of  the  single  amino-acid  Lysine.  But  upon  readmission  of  lysine  to 
the  diet,  even  at  an  age  exceeding  the  average  normal  duration  of  life 
(700  days),  growth  is  immediately  inaugurated,  at  the  same  speed 
that  it  would,  in  the  normal  course  of  events,  have  taken  place  in  a 
normally  fed  animal  of  similar  weight  and  stage  of  development. 
The  retardation  of  growth  by  the  accumulation  of  the  products  of 
growth  is  therefore  one  of  the  important  factors  in  determining  the 
inability  of  the  adult  tissues  to  maintain  their  weight  in  aged  animals. 
It  is  not  the  only  factor,  however,  because  in  that  case,  as  we  have 
seen,  indefinitely  prolonged  equilibrium  and  not  decline  would  be  the 
resultant. 

A  modification  of  Butschli's  hypothesis  is  that  proposed  by  Rubner, 
namely,  that  the  protoplasm  of  an  animal  is  able  to  sustain  a  limited 
number  of  molecular  transformations  and  no  more.    Thus  he  points 
33 


514    PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

out  that  the  total  calorific  output  of  a  variety  of  animals  from  birth 
to  old  age  is  approximately  the  same,  a  striking  exception,  being, 
however,  afforded  by  man: 
TOTAL  CONSUMPTION  OF  CALORIES  PER  KILOGRAM  OF  BODY^-WEIGHT. 

Man 725,770 

Horse •  169,900 

Cow 141,090 

Dog 163,900 

Cat 223,800 

Guinea-pig 265,500 

The  instances  are,  however,  not  very  numerous  and  if  one  marked 
exception  to  the  "rule"  occurs  among  such  a  small  number  of  cases, 
other  exceptions  will  doubtless  be  encountered.  Indeed  we  may 
with  more  probability  attribute  the  exceptional  position  of  man  in 
this  small  group  to  the  much  larger  proportion  of  Nervous  Tissues; 
tissues,  that  is,  of  high  metabolic  rate,  which  his  body  contains  in 
comparison  with  the  other  animals  enumerated.  His  duration  of  life 
is  also,  and  possibly  for  the  same  reason,  exceptionally  great. 

Quite  a  different  type  of  hypothesis  to  the  foregoing  is  that  pro- 
posed by  Metchnikoff,  who  attributes  senescence  in  part  to  the  aber- 
rant activities  of  Phacocytes  and  in  part  to  the  absorption  of  toxic 
substances  which  are  products  of  bacterial  decomposition  in  the  lower 
intestine.  While  there  can  be  little  doubt  that  some  of  the  tissue- 
changes  which  are  characteristic  of  old  age,  such  as  sclerosis,  vascular 
lesions  and  so  forth  may  be  hastened  or  even  brought  about  by  repeated 
administrations  of  basic  substances,  such  as  Adrenaline  or  Tyramine 
which  may  be  derived  from  amino-acids  by  Decarboxylation,  yet  as 
a  general  hypothesis  of  senescence  this  is  too  specific,  too  limited  in  its 
scope  and  applicability,  to  account  for  the  phenomenon  in  the  multi- 
tude of  the  forms  of  life  which  exhibit  it.  In  fact,  Metchnikoff  did 
not  advance  his  hypothesis  as  an  explanation  of  "natural"  old  age, 
although  he  is  commonly  accredited  with  having  done  so,  but  as  an 
explanation  of  what  he  considered  to  be  the  "premature"  senescence 
of  human  beings,  and,  as  such,  it  is  a  hypothesis  which  deserves  very 
serious  consideration.  The  effects  produced  by  basic  Nitrogenous 
Poisons  related  to  the  amino-acids  are,  however,  confined  to  certain 
tissues  and  especially  the  circulatory  and  renal  systems,  while  the 
effects  of  senescent  atrophy  modify  in  greater  or  less  degree  every 
tissue  in  the  body.  Organisms  in  which  the  structural  changes  pro- 
ducible by  poisons  of  this  character  could  not  constitute  an  irreparable 
injury  nevertheless  display  senescence  and  its  necessary  outcome, 
"natural  death." 

The  unicellular  animals  and  certain  unorganized  types  of  living 
tissue,  such  as  cancer-tissue,  are,  as  Wiessmann  and  Loeb  have  espe- 
cially emphasized,  actually  or  potentially  immortal.1  The  Unicellular 

Those  forms  which  undergo  periodical  conjugation  may  also  exhibit  senescence, 
which,  however,  may  very  possibly  be  due  to  causes  analogous  to  those  described  below 
which  lead  to  senescence  in  the  metazoa.  Cf.,  G.  N.  Calkins:  Proc.  Soc.  Exper.  Biol. 
and  Med.,'1919,  16,  p.  57, 


OLD  AGE  AND  SENESCENCE  515 

Organisms  subdivide,  and  the  daughter-cells  which  thus  arise  each 
contain  the  protoplasm  of  the  parent-cell  which  is  thus  perpetuated 
indefinitely.  No  slackening  of  the  process  of  reproduction  occurs 
unless  the  supply  of  nutrients  fails.  Even  in  those  forms  such  as  the 
Infusoria,  in  which  conjugation  of  two  cells  occasionally  occurs,  this 
is  not  generally  essential  to  the  maintenance  of  the  indefinite  repro- 
ducibility  of  the  original  protoplasm.  In  the  growth  of  Cancer  only 
the  failure  of  the  tissues  of  the  host  to  support  the  parasitic  tissue 
sets  a  term  to  its  existence.  If  the  tissue  be  transplanted  from  time 
to  time  into  a  fresh  host  it  is  propagated  indefinitely.  The  failure  of 
nutrients  is  again  the  only  factor  which  limits  indefinite  reproduction. 

The  mortality  of  higher  organisms  is  therefore  a  consequence  of 
their  complexity,  and  a  very  probable  explanation  lies  in  the  sub- 
division and  delegation  of  functions  and  powers  which  renders  this 
complexity  possible.  There  is  a  very  noticeable  alteration  in  the 
relative  proportions  of  the  different  types  of  tissue  in  the  body  with 
advancing  age.  As  Metchnikoff  has  expressed  it:  "Old  age  is  char- 
acterized by  a  conflict  between  the  finer  and  more  complicated  ele- 
ments and  the  simple  or  more  primitive  elements  of  the  organisms, 
a  conflict  that  ends  to  the  advantage  of  the  latter.  The  picture  is 
always  the  same — atrophy  of  the  more  highly  differentiated  elements 
and  their  replacement  by  an  overgrowth  of  connective  tissue."  In 
other  words  Sclerous  Tissues  acquire  a  dominance  over  the  Parenchy- 
matous  Tissues  which  are  the  most  important  or  perhaps  exclusive 
source  of  the  endogenous  catalyzers  of  growth. 

The  senescent  decay  of  the  body  may,  in  fact,  be  attributable  to 
the  increasing  mass  of  dependent  tissues  with  which  nutrients  must 
be  shared  and  for  the  production  and  repair  of  which  catalyzers 
must  be  provided.  So  long  as  the  velocity  of  the  forward  reaction  of 
growth  predominates  sufficiently  over  that  of  the  backward  reaction, 
the  impulse  to  growth  secures  the  continued  accretion  of  tissue.  Part 
of  this  tissue  assists  in  the  production  of  catalyzers,  but  part,  that 
part  constituted  by  the  tissues  of  structural  rather  than  functional 
significance,  merely  draws  away  nutrients  from  the  tissues  which 
produce  the  endogenous  catalyzers.  This  has  the  effect,  so  far  as  the 
self-maintaining  tissues  are  concerned,  of  progressive  reduction  of  the 
Nutrient-le-vel,  or  diminution  of  the  value  of  aa"  in  the  autocatalytic 
equation.  The  value  of  aa,"  however,  determines  the  ultimate  or 
equilibrium-weight  of  the  animal  and  as  it  sinks  so  must  the  weight 
of  the  animal  diminish,  the  parenchymatous  tissues  being  directly 
and  the  sclerous  tissues  only  indirectly  affected.  Hence  the  proportion 
of  sclerous  to  parenchymatous  tissues  is  further  enhanced  and  the 
process  of  senescence  itself  partakes  of  the  autocatalytic  character. 
•  It  should  be  especially  noted  in  this  connection  that  the  cost  of 
production  of  Sclerous  Tissues  is  not  to-be  estimated  merely  in  terms 
of  their  mass.  They  are  "expensive"  tissues  to  manufacture  in  com- 
parison with  the  parenchymatous  tissues.  Not  only  are  they  poorer  in 


516     PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

water  and  therefore  richer  in  organic  materials  than  the  parenchy- 
matous  tissues,  but  the  Proteins  which  they  contain  are  of  very  abnor- 
mal composition,  a  composition  which  is  specific  for  each  type  of 
sclerous  tissue.  They  are  incomplete  proteins,  containing  certain 
ammo-acid  radicals  in  exceptional  abundance,  while  others  which 
usually  occur  in  proteins  of  cellular  origin  are  lacking  or  present  in 
unusually  small  amounts.  To  manufacture  one  molecule  of  a  protein 
of  this  abnormal  character  several  molecules  of  the  ordinary  types  of 
protein  must  be  sacrificed,  just  as  several  buildings  constructed  of 
wood,  stone  and  brick  must  be  sacrificed  to  obtain  the  materials 
wherewith  to  construct  a  similar  building  entirely  of  stone  or  of  brick. 
Hence  the  drain  upon  the  nutrient-level  in  the  circulating  fluids  which 
is  brought  about  by  the  sclerous  tissues  is  far  more  than  proportionate 
to  their  mass. 

We  have  seen  that  the  administration  of  Growth-catalyzers  must 
favor  the  development  of  parenchymatous  as  opposed  to  sclerous 
tissues.  Corresponding  with  this  view  and  with  the  views  expressed 
above  concerning  the  origin  of  senescence,  we  find  that  the  continuous 
administration  of  Tethelin  to  mice,  from  the  fifth  week  of  age  onward, 
or  even  its  intermittent  administration  for  several  brief  periods,  leads 
to  a  remarkable  prolongation  of  the  average  Duration  of  Life.  Thus 
the  duration  of  life  of  normal  white  mice  was  found  in  the  particular 
stock  employed  to  be  767  days  for  males  and  719  days  for  females 
within  a  probable  error  of  somewhat  less  than  one  month.  Males 
which  had  received  4  mgm.  of  tethelin  daily  throughout  their  lives 
attained  an  average  age  of  866  days  before  death,  while  females  inter- 
mittently receiving  the  same  dosage  attained  an  average  age  of  800 
days.  This  would  be  equivalent  to  a  prolongation  of  from  ten  to 
fifteen  years  in  the  average  duration  of  life  in  man.  Pituitary  (anterior 
lobe)  tissue,  cholesterol,  and  lecithin  alike  failed  to  influence  the 
duration  of  life,  the  pituitary  tissue  on  account  no  doubt  of  the  small- 
ness  of  the  dosage  of  tethelin  contained  in  the  amount  of  the  tissue 
which  it  was  practicable  to  administer,  and  cholesterol  on  account  of 
the  secondary  deleterious  effects  of  the  deposits  of  this  substance 
which  accumulate  in  the  tissues  of  animals  receiving  excessive  amounts. 
The  absence  of  any  effect,  of  the  administrations  upon  the  life-duration 
of  these  various  groups  of  animals  rendered  them  additional  "controls" 
by  reference  to  which  the  prolongation  of  life  attained  by  the  adminis- 
tration of  tethelin  could  be  gauged.  The  average  duration  of  life  of 
the  tethelin-fed  males  was  found  to  exceed  the  average  life-duration 
of  the  males  of  all  other  classes  of  animals  investigated  by  one  hundred 
and  three  days,  while  the  life-duration  of  the  tethelin-fed  females  ex- 
ceeded that  of  all  other  classes  by  one  hundred  and  eight  days.  The 
chance  of  both  of  these  deviations  from  normality  being  "accidental" 
was  computed  to  be  only  1  in  1 1,000.  The  prolongation  of  life  in  mice 
by  the  continuous  or  frequent  administration  of  relatively  large  doses 
of  tethelin  is  therefore  unmistakable,  Furthermore,  Senescence  is  very 


OLD  AGE  AND  SENESCENCE 


517 


much  delayed  in  tethelin-fed  animals,  the  loss  of  weight  for  a  prolonged 
period  being  almost  imperceptibly  gradual,  whereas  in  normal  animals 
it  is  relatively  sudden  (Fig.  41,  p.  506). 

We  have  seen  that  the  tissues  of  the  Nervous  System  are  very  rich 
in  lipoids  which  are  either  identical  with  (cholesterol)  or  related  to 
(phospholipins,  etc.),  the  substances  which  we  know  to  have  an 
influence  upon  growth  similar  to  that  which  we  would  expect  to  be 
exerted  by  catalyzers  of  growth.  Furthermore  their  exceptionally 
high  Metabolic  Rate  encourages  the  supposition  that  they  produce  an 
abundance  of  endogenous  growth-catalyzers.  A  predominant  develop- 
ment of  nervous  tissues  should  therefore  be  equivalent  in  its  effects 
upon  metabolism,  growth,  and  life-duration  to  the  continuous  adminis- 
tration of  an  excess  of  growth-catalyzers. 

Now  Friedenthal  has  pointed  out  that  the  ratio  of  brain-weight  to 
body-weight  or  to  the  two-thirds  power  of  the  body-weight,  which 
he  terms  the  "  cephalization-f actor,"  varies  from  one  species  of  animal 
or  bird  to  another  in  extremely  close  correspondence  with  the  maximal 
attainable  duration  of  life.  The  following  are  among  the  figures  which 
he  cites  in  support  of  this  thesis:1 

MAMMALS. 

Maximal  life-duration 

Cephalization-factor.  (according  to  Hanseman 

Species.  in  years. 

Man.      . 2. 67  to  2. 81  80  to  150 

Elephant 1.24  to  1.34  90  to  100  • 

Anthropoid  apes 0.76  to  0.65  

Horse I,   ...  0.43  to  0.57  45 

Deer v.     ..  0.40to0.50  30 

Bears 0.36  to  0.50  50 

Dogs 0.34  to  0.51  15  to  20 

Cats 0.29  to  0.34  20 

Oxen          1 

Giraffes      >         0.30  to  0.40  30 

Antelopes  J 

Squirrels 0.16  to  0.20  6 

Insectivora 0.06  to  0.18  6  to  10 

Mice ..."           0.04  3 

BIRDS. 

Maximal  life-duration 
(according  to  Hansemann 
Species.  Cephalization-factor.  in  years. 

Carrion  crow 0.168  100  (?) 

Parrots 0.147  to  0.177  100  (?) 

Alpine  crow 0.114  50 

Buzaard 0.11 

Owl 0.113 

Finch 0.086  8 

Sparrow 0.086 

Duck 0.0731 

Snipe 0.0585 

Quail 0.0495 

Heron .  0.0459  15 

Pheasant .  0.0343  15 

Fowls 0.0249  10  to  20 

Ostrich    .  0. 0195 


1  The  life-duration  of  the  mouse  computed  from  the  observations  cited  above  has 
been  added  to  the  table. 


518    PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

The  various  estimates  of  the  maximal  Duration  of  Life  can  only  he 
regarded,  excepting  in  the  case  of  the  mouse,  as  very  hazardous 
approximations,  since,  even  in  the  case  of  man,  the  maximal  attainable 
duration  of  life  has  been  the  subject  of  far  more  fables  than  investi- 
gations. Probably  the  mean  duration  of  life  would  be  a  better  standard 
of  comparison  than  the  maximal  duration  of  life,  since  the  magnitude 
of  the  latter  estimate  may  be  so  greatly  affected  by  a  single  exceptional 
observation.  On  the  other  hand  statistical  estimates  of  the  average 
duration  of  life  are  lacking,  save  for  man  and  mice,  and  even  the 
estimates  for  man  which  are  available  include  accidental  deaths  and 
deaths  from  epidemic  infections.  However,  notwithstanding  the 
approximate  character  of  the  estimates,  they  afford  very  striking  evi- 
dence of  a  tendency  of  Longevity  to  be  associated  with  a  high  degree  of 
development  of  the  nervous  system.  Thus,  so  far  as  the  effect  upon 
the  duration  of  life  is  concerned,  exceptional  development  of  the 
nervous  system  exerts  an  effect  similar  to  that  which  is  induced  by 
the  administration  of  an  excess  of  a  growth-catalyzer. 

The  resemblance  between  the  effects  of  a  high  proportion  of  Nervous 
Tissues  and  those  induced  by  administration  of  a  growth-catalyzer 
extends,  however,  even  to  the  time-relations  of  growth,  as  expressed 
by  the  contours  of  the  growth-curve.  Thus  on  comparing  the  growth- 
curves  for  man  and  mice  in  Fig.  31  (p.  473),  with  the  growth-curves  for 
cholesterol-fed  and  tethelin-fed  mice  in  Figs.  40  and  41  (pp.  505  and 
506),  it  is  at  once  apparent  that  the  change  in  the  time  relations  of 
the  growth  of  mice  which  is  induced  by  these  catalyzers  brings  their 
growth-curve  into  close  approximation  to  the  human  curve.  The  effect 
of  the  growth-catalyzers  in  unusual  amount  is  to  apparently  prolong 
the  second  and  abbreviate  and  accelerate  the  third  growth-cycle,  and 
it  is  in  precisely  these  characteristics  that  the  human  growth-curve, 
when  reduced  to  the  same  scale,  differs  most  strikingly  from  the 
growth-curve  for  mice.  It  is  not  unlikely,  therefore,  that  the  differ- 
ence in  contour  of  the  mouse  and  human  curves  of  growth  is  attribu- 
table to  the  greater  abundance  of  endogenous  catalyzers  of  growth  in 
the  tissues  and  tissue-fluids  of  man  consequent  upon  the  greater  pro- 
portionate development  of  his  nervous  system. 

REFERENCES 

GENERAL  CHARACTERISTICS  OF  THE  GROWTH-PROCESS: 
Voit,  C.:     Zeit.  f.  Biol.,  1866,  2,  p.  353. 
Bowditch:     Eighth  Annual  Report,  State  Board  of  Health,  Massachusetts,  U.  S.  A., 

1877. 

Roberts:     Manual  of  Anthropometry,  London,  1878. 

Anthropometric  Committee,  British  Assn.  Reports,  1879,  p.  175;  1883,  p.  253. 
Minot:     Jour.  Physiol.,  1891,  12,  p.  97. 
Porter:    Trans.  Acad.  Sci.,  St.  Louis,  1895,  6,  p.  263. 
Voit,  E.:     Zeit.  f.  Biol.,  1905,  46,  p.  195. 
Donaldson:     Boas  Memorial  Volume,  New  York,  1906,  p.  5.     The  Rat,  Pub.  of  the 

Wistar  Institute,  Philadelphia,  1915. 

Loeb,  J.:     Seventh  Internat.  Zool.  Congress,  Boston,  1907. 
Ostwald:     Vortrage  und  Aufsatze  iiber  Entwicklungsmech.,  Leipzig,  1908,  Heft  5. 


OLD  AGE  AND  SENESCENCE 

GENERAL  CHARACTERISTICS  OF  THE  GROWTH-PROCESS: 

Robertson:  Arch.  f.  Entwioklungsmech.,  1908,  25,  p.  581;  1908,  26,  p.  108;  1913, 
37,  p.  497.  Biol.  Centr.,  1910,  30,  p.  316;  1913,  33,  p.  29.  Am.  Jour.  Physiol., 

1915,  37,  pp.  1  and  74;  1916,  41,  pp.  535  and  547.     Tables  for  the  Computation 
of  Curves  of  Autocatalysis,  with  Special  Reference  to  Curves  of  Growth,  Univ. 
California  Pubs.  Physiol.,   1915,  4,  p.   211. 

Stratz:     Der  Korper  des  Kindes  und  seine  Pflege,  Stuttgart,  1909,  3.  aufl.  . 

Read:     Arch.  f.  Entwicklungsmech.,  1913,  35,  p.  708. 

Martin:  Lehrbuch  der  Anthropologie,  Jena,  1914  (consult  for  literature  con- 
cerning the  growth  of  man). 

Thompson:     Growth  and  Form,  London,  1917. 
SUBSTRATES  OF  GROWTH: 

Hopkins  and  Willcock:     Jour.  Physiol.,  1906-7,  35,  p.  88. 

Mendel  and  Mitchell:     Am.  Jour.  Physiol.,  1907,  20,  p.  81. 

Mendel  and  Saiki:     Ibid.,  1908,  21,  p.  64. 

Stepp:  Biochem.  Zeit.,  1909,  22,  p.  452.  Zeit.  f.  Biol.,  1912,  57,  p.  135;  1912,  59, 
p.  366;  1913,  62,  p.  405. 

Osborne  and  Mendel:  Science,  N.  S.,  1911,  34,  p.  722;  1917,  45,  p.  294.  Jour. 
Biol.  Chem.,  1912,  12,  p.  81;  1912-13,  13,  p.  233;  1913-14,  16,  p.  423;  1914,  18, 
p.  95;  1917,  31,  p.  149.  Am.  Jour.  Physiol.,  1916,  40,  p.  16.  Biochem.  Jour., 

1916,  10,  p.  534. 

Funk:     Jour.  Physiol.,  1911-12,  43,  p.  395;  1912,  44,  p.  50.     Ergeb.  d.  Physiol., 

1913,  13,  p.  125.     Jour.  Biol.  Chem.,  1916,  27,  p.  1. 
Hopkins:     Jour.  Physiol.,  1912,  44,  p.  425. 
McCollum  and  Davis:     Jour.  Biol.  Chem.,  1913,  15,  p.  167;  1914,  19,  pp.  245  and 

323;  1915,  20,  pp.  415  and  641;  1915,  23,  pp.  181  and  231.     Jour.  Am.  Med. 

Assn.,  1917,  68,  p.  1379. 
Hart  and  McCollum:     Ibid.,  1914,  19,  p.  373. 

Mendel:     Nutrition  and  Growth,  Harvey  Lectures,  1914-15,  10,  p.  101. 
McCollum:     Supplementary  Relationships  among  our  Natural  Foodstuffs,  Harvey 

Lecture,   1915-16,  11,  p.   151. 

Funk  and  Macallum:     Jour.  Biol.  Chem.,  1915,  23,  p.  413;  1916,  27,  p.  51. 
Macallum:     American  Medicine,  new  series,  1916,  11,  p.  782. 
RELATIONSHIP  OF  THE  ENDOCRINE  ORGANS  TO  GROWTH: 

Gushing:     The  Pituitary  Body  and  its  Disorders,  Philadelphia,   1912. 

Aldrich:     Am.  Jour.  Physiol.,  1912,  30,  p.  352;  1912-13,  31,  p.  94. 

Schafer:     Quar.  Jour.  Exp.  Physiol.,  1912,  5,  p.  203. 

Gudernatsch:     Am.  Jour.  Anat.,  1913-14,  15,  p.  431. 

Wulzen:    Am.  Jour.  Physiol.,  1914,  34,  p.  127. 

Adler:     Arch.  f.  Entwicklungsmech.,  1914,  39,  p.  21. 

McCord:     Interstate  Medical  Jour.,  1915,  22,  p.  354. 

Pearl:     Jour.  Biol.  Chem.,  1916,  24,  p.  123. 

Robertson:     Ibid.,  1916,  24,  p.  385. 

Smith:     Anat.  Record,  1916-17,  11,  p.  410.    University  of  California  Pubs.,  Physiol., 

1918,  5,  p.  11. 

Uhlenhuth:     Jour.  Gen.  Physiol.,  1918-19,  1,  pp.  23,  33,  305,  315,  473,  525. 
Robertson  and  Ray:     1919,  37,  pp.  393,  427,  443. 
OLD  AGE  AND  SENESCENCE: 

Metchnikoff:     The  Nature  of  Man,  New  York,  1903.     The  Prolongation  of  Life, 

New  York,  1910. 

Minot:     The  Problem  of  Age,  Growth  and  Death,  New  York,  1907. 
Adami:     Principles  of  Pathology,  Philadelphia,  1908,  1,  p.  125. 
Rubner:     Kralt  und  Stoff  im  Haushalte  der  Natur,  Leipzig,  1909. 
Friedenthal:     Contr.  f.  Physiol.,  1910,  24,  p.  321. 
Woodruff:     Arch.  f.  Protistenkunde,  1910-11,  21,  p.  263. 
Fleischer  and  Loeb,  L.:     Proc.  Soc.  Exp.  Biol.  and  Med.,  1911,  8,  p.  133. 
Saundby:     Old  Age;  Its  Care  and  Treatment  in  Health  and  Disease,  London,  1914. 
Child:     Senescence  and  Rejuvenescence,  Chicago,  1915. 
Loeb,  J.:     The  Organism  as  a  Whole,  New  York,  1916. 
Cramer:     Jour.  Physiol.,  1916,  50,  p.  322. 
CATALYZERS  OF  GROWTH  : 

King:     Biol.  Bull.,  1907,  13,  p.  40. 

Loeb,  L.:     Jour.  Am.  Med.  Assn.,  1908,  50,  p.  1897;  1909,  53,  p.  1471.     Arch.  f. 

Entwicklungsmech.,  1909,  27,  p.  89;  1911,  32,  pp.  67  and  662. 
Johnson:     Univ.  California  Pubs.,  Zoology,   1913,  11,  p.  53. 


520     PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

CATALYZERS  OF  GROWTH: 

Bain:     Lancet,  1913,  182,  p.  918. 

Browder:     Univ.  California  Pubs.,  PhysioL,  1915,  5,  p.  1. 

Robertson  and  Cutler:     Jour.  Biol.  Chem.,  1916,  25,  p.  663. 

Robertson  and  Ray:     Ibid.,  1916,  24,  p.  347;  1919,  37,  pp.  377,  393,  427,  443. 

Robertson:     Ibid.,  1916,  24,  pp.  363,  385,  397,  409;  1916,  25,  pp.  635,  647. 

Robertson  and  Delprat:     Ibid.,  1917,  31,  p.  567. 

Robertson:     Endocrinology,  1917,  1,  p.  24. 
R6LE  OF  CATALYZERS  IN  CANCER: 

Wacker:     Zeit.  f.  Physiol.  Chem.,  1912,  80,  p.  383. 

Burnett:     Proc.  Soc.  Exp.  Biol.  and  Medicine,  1913-14,  11,  p.  42. 

Robertson  and  Burnett:     Jour.  Exp.  Med.,  1913,  17,  p.  344;  1915,  21,  p.  280;  1916, 
23,  p.  631.     Proc.  Soc.  Exp.  Biol.  and  Med!,   1913,  10,  pp.  140  and  143. 

Bennett:     Jour.  Biol.  Chem.,  1914,  17,  p.  13. 

Sweet,  Corson-White  and  Saxon:     Ibid.,  1915,  21,  p.  309. 

Robertson  and  Burnett:     Jour.  Cancer  Research,  1918,  3,  p.  75. 

Luden:   Jour.  Lab.  and  Clin.  Med.,  1916,  1,  p.  662;  1917,  3,  pp.  93  and  141;  1918-19, 

4,  p.  849.     Jour.  Biol.  Chem.,  1916,  27,  p.  273;  1917,  29,  p.  463. 
HEALING  OF  WOUNDS: 

Carrel:     Jour.  Am.  Med.  Assn.,  1910,  55,  p.  2148. 

Robertson:    Ibid.,  1916,  66,  p.  1009. 

Spain  and  Loeb,  L.:     Jour.  Exp.  Med.,  1916,  23,  p.  107. 

Carrel  and  Hartman:     Ibid.,  1916,  24,  p.  429. 

Du  Nouay:     Ibid.,  1916,  24,  p.  451;  1917,  25,  p.  721. 

Barney:     Jour.  Lab.  and  Clin.  Med.,  1918,  3,  p.  480. 

Clark:    Bull.  Johns  Hopkins  Hospital,  1919,  30,  p.  117. 


CHAPTER  XXI. 

PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION: 
MEMORY  AND  SLEEP. 

MEMORY. 

THE  most  prominent  characteristic  of  the  Nervous  System  is  the 
facilitation  of  its  functions  which  their  performance  brings  about. 
A  mental  task  which  is  at  first  difficult  becomes  easy  by  frequent  repe- 
tition; an  act  which  may  be  performed  under  the  guidance  of  the 
central  nervous  system  at  first  only  with  effort  and  concentration  of 
the  will  and  attention,  becomes  by  repetition  a  habit  or  even  a  reflex 
which  is  performed  almost  automatically  and  without  any  conscious 
expenditure  of  effort. 

Secondary  and  subsequent  to  this  phenomenon  of  facilitation  is  the 
phenomenon  of  Fatigue.  For  example,  in  the  learning  of  a  long  passage 
by  rote,  as  one  tries  to  recall  it  after  the  first  repetition,  recollection  is 
distinctly  difficult.  With  a  second  repetition  recollection  is  easier, 
with  a  third  it  is  easier  still  and  so  the  progressive  facilitation  accumu- 
lates until  it  becomes  possible  to  repeat  a  long  passage  from  "Memory," 
faultlessly  and  fluently.  If,  however,  the  repetitions  be  still  continued 
or  fresh  matter  added  to  the  lesson  a  new  phenomenon  supervenes 
which  is  the  reverse  of  that  initially  experienced.  The  passage  which  a 
little  while  before  was  repeated  faultlessly  cannot  now  be  repeated 
without  mistakes.  The  attention  wanders  readily.  Recollection 
becomes  increasingly  difficult,  the  consciousness  has  to  be  "flogged' ' 
into  activity  and  finally  excessive  fatigue  compels  desistance  from  the 
task.  The  effects  of  the  initial  facilitation  have  not  been  undone, 
however,  for  a  return  to  the  task  after  an  adequate  interval  for  recuper- 
ation reveals  the  fact  that  the  previous  study  has  implanted  memories 
which  disappear  from  the  field  of  consciousness  in  many  instances 
only  after  a  lapse  of  time  comparable  with  the  duration  of  life  itself. 

We  meet,  therefore,  in  the  exercise  of  any  given  intellectual  function, 
with  two  apparently  contradictory  facts.  Performance  facilitates  the 
exercise  of  the  function,  and  it  likewise  depresses  the  exercise  of  the 
function.  We  note,  furthermore,  that  the  facilitation  and  depression 
become  evident  at  different  periods  of  time,  the  former  in  the  earlier 
stages  of  performance  and  the  latter  in  its  later  stages. 

Many  hypotheses  have  been  advanced  by  philosophers,  psychologists 
and  physiologists  in  the  endeavor  to  imagine  a  mechanism  which  could 
account  for  the  phenomenon  of  memory.  The  vast  majority  of  the 
mechanistic  hypotheses,  which  are  the  only  ones  of  which  we  need 


522    PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

attempt  the  consideration,  partake  of  the  same  general  character; 
they  assume  that  the  previous  repetition  or  performance  has  left  some 
species  of  more  or  less  permanent  modification  in  the  nervous  system, 
and  they  vary  only  in  the  nature  of  this  hypothetical  modification. 

Broadly  speaking,  the  nature  of  this  modification  may  be  conceived 
in  either  of  two  ways  which,  for  convenience  sake,  we  may  designate, 
respectively,  the  "static  modification"  and  the  "dynamic  modifi- 
cation." The  static  conception,  as  developed  especially  by  Munk 
and  Ziehen,  regards  the  "trace"  or  "image,"  which  has  been  formed 
in  the  nervous  system  in  consequence  of  some  act  or  repetition,  as 
consisting  of  some  structural  modification,  some  physical  alteration, 
an  alteration,  in  other  words,  in  the  distribution  of  cell-matter  in  space. 
The  objections  which  may  be  and  have  been  urged  against  this  view 
are  manifold.  A  purely  physical  alteration,  namely  the  redistribution 
of  preformed  cell-material  in  space,  would  be  something  of  the  nature 
of  a  strain  produced  in  response  to  some  stress  (=  stimulus)  which 
might  be  conceived  of  as  mechanical,  electrical,  thermal  or  yet  some 
other  type  of  energy-change  capable  of  inducing  modifications  of  the 
physical  state  of  matter.  Now  the  remarkable  Persistence  of  Memories 
proves  that  the  "trace,"  whatever  it  may  be,  is  rather  permanent  and 
only  very  slowly  fades  away.  Indeed  such  investigations  as  those  of 
Prince  or  Sidis  would  appear  to  indicate  that  a  large  proportion  of 
memory-traces  may  persist  in  some  measure  throughout  a  lifetime. 
Of  course,  reinforcement  of  the  trace  by  occasional  "recollection," 
either  conscious  or  "subconscious"  may  have  occurred  from  time  to 
time  in  the  interval  between  the  receipt  of  an  impression  and  its  emer- 
gence from  consciousness  under  abnormal  psychological  conditions, 
such  as  those  imposed  by  Hypnosis,  at  a  much  later  period  of  life. 
Reinforcement  of  the  trace  by  recollection  cannot,  however,  be  the 
general  rule,  for  otherwise,  as  Sidis  has  pointed  out,  our  entire  mental 
life  would  be  occupied  in  recollecting. 

The  memory  trace,  or  at  least  some  residual  fragment  of  it,  is  there- 
fore an  extraordinarily  persistent  modification.  The  material  of  which 
the  central  nervous  system  is  composed,  however,  is  largely  fluid  or 
semifluid,  and  all  our  experience  teaches  as  that  a  fluid  cannot  retain 
physical  strains  for  any  prolonged  period;  indeed  it  is  this  quality 
which  enables  us  to  recognize  a  fluid  or  a  jelly  and  distinguish  it  from 
a  solid. 

A  modification  of  the  theory  of  Munk  is  that  which  was  proposed 
by  Lepine  and  Duval  and  has  been  very  widely  adopted  by  a  certain 
school  of  neurologists  and  psychologists.  This  theory  is  based  upon 
the  demonstration  by  Cajal  that  the  nervous  system  is  divided,  like 
other  tissues,  into  distinct  cell-units,  or  Neurons,  which  he  regarded 
as  being  in  contact  with  one  another  through  the  medium  of  their  cell- 
processes  or  Dendrites,  but  not  physically  continuous  with  one  another. 
It  was  assumed  by  Lepine  that  the  formation  of  a  new  memory-trace 
in  the  nervous  system  was  attributable  to  the  formation  of  a  new  den- 


MEMORY  523 

driteHL'ontact,  while  Amnesia  or  the  phenomenon  of  forgetting  repre- 
sents the  breaking  of  a  contact  previously  established.  To  this  view 
there  attach  most  of  the  difficulties  attendant  upon  Munk's  hypothesis 
and,  furthermore,  as  Meyer  has  very  justly  pointed  out1  the  invo- 
cation of  such  hypothetical  structural  changes  to  explain  the  physical 
correlates  of  psychic  phenomena  must  necessarily  lead,  sooner  or  later, 
to  the  invention  of  a  metaphysical  entity  to  keep  the  apparatus  in 
order.  Meyer  expresses  this  difficulty  as  follows :  "  Why  does  the  pro- 
toplasm stretch  toward  one  neighboring  neurone  when  the  organism 
happens  to  be  in  one  situation,  toward  another  neurone  when  the  organ- 
ism is  in  another  situation?  General  silence  with  the  neurologists. 
But  some  psychologists  had  an  answer  ready.  They  brought  in  their 
deus  ex  machina.  The  Ghost  does  it.  Consciousness,  feeling,  will, 
or  whatever  you  call  it,  turns  the  bridge  in  the  proper  direction  as  the 
switchman  turns  the  switch  in  a  railway-yard."  The  cytological  basis 
of  this  hypothesis  has  also  been  called  severely  in  question  since  the 
investigations  of  Epathy,  Bethe  and  others  have  demonstrated  the 
existence  of  fine  intercommunicating  fibrils  which,  in  many  instances 
at  least,  establish  anatomical  continuity  between  adjacent  dendrites. 

The  dynamic  conception  of  the  memory-trace,  on  the  other  hand, 
regards  it  as  being  formed  by  a  chemical  alteration  of  cell-material 
along  the  nervous  path  which  was  followed  by  the  stimulus  which  is 
subsequently  recalled.  The  superior  generality  and  simplicity  of  this 
hypothesis  is  evident  at  once.  It  does  not  exclude  the  possible  forma- 
tion of  a  definite  structure  as  the  result  of  chemical  change,  on  the 
other  hand  the  persistence  of  memory  traces  is  at  once  accounted  for 
since,  as  we  have  abundant  reason  to  know,  chemical  changes  within 
living  organisms  may  be  as  enduring  as  life  itself. 

We  have  seen  (Chapter  XVIII)  that  the  rate  of  conduction  of 
impulses  in  Nerve-fibers  is  conditioned  partly  if  not  wholly  by  physical 
changes  which  underlie  the  passage  of  the  impulse.  We  infer  this  from 
the  low  Temperature-coefficient  of  conduction  in  peripheral  nerve- 
fibers.  In  Nerve-cells,  on  the  contrary,  the  passage  of  impulses  is 
demons trably  accompanied  by  chemical  changes.  The  temperature- 
coefficient  for  the  conduction  of  impulses  in  the  nerve-cells  of  the 
respiratory  center  and  the  cardiac  ganglion,  for  example,  is  of  the 
chemical  order  of  magnitude.  Furthermore,  as  Mosso  has  demon- 
strated, excitation  of  the  cerebral  cortex  results  in  a  pronounced 
disengagement  of  heat.  Repeated  attempts  to  demonstrate  a  similar 
evolution  of  heat  in  nerve-fibers  in  consequence  of  stimulation  have 
failed.  The  processes  which  attend  the  conduction  of  impulses  through 
nerve-cells,  therefore,  appear  to  be  of  a  fundamentally  different  char- 
acter from  those  which  accompany  the  passage  of  impulses  in  nerve- 
fibers. 

The  effect  of  the  chemical  change  which  accompanies  the  passage  of 

1  Meyer:  Journal  of  Philos.  Psychol.  and  Scientific  Methods,  1912,  9,  p.  365. 


524    PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

an  impulse  through  the  central  nervous  system  is  to  initially  facilitate 
and  ultimately  retard  the  passage  of  subsequent  impulses  along  the 
same  path.  The  nature  of  the  initial  facilitation  has  been  variously 
characterized.  Thus  Maudsley  described  it  as  the  formation  of  a 
trace  or  thread  of  a  deposit  which  is  followed  by  the  succeeding  impulse, 
while  Exner  likened  it  to  the  "excavation  of  a  channel/'  a  hypothesis 
which  is  generally  referred  to  as  the  Canalization  Hypothesis. 

In  preceding  chapters  we  have  had  frequent  occasion  to  dwell  upon 
a  variety  of  chemical  processes  and  not  a  few  life-phenomena  which 
display  initial  facilitation  followed  by  retardation.  These  are  the 
various  processes  or  phenomena  which  are  governed  as  to  their  speed 
by  underlying  Autocatalyzed  Reactions.  It  is  evident  that  if  the  passage 
of  an  impulse  through  the  central  nervous  system  were  attributable  to 
the  occurrence  of  an  autocatalyzed  chemical  reaction,  the  deposition 
of  the  products  of  this  reaction  along  the  path  of  the  impulse  would 
facilitate  the  passage  of  a  subsequent  impulse,  while  their  accumula- 
tion in  undue  amount  would  constitute  an  impediment  to  the  further 
occurrence  of  the  reaction  and  therefore  to  the  passage  of  subsequent 
impulses.  The  same  mechanism  thus  accounts  for  both  the  facilitation 
and  the  fatigue  which  accompany  the  performance  of  functions  involv- 
ing the  central  nervous  system. 

Regarding  the  nature  of  the  autocatalyst  in  this  reaction  we  are  of 
course  completely  in  the  dark  in  so  far  as  any  direct  results  of  chemical 
analysis  are  concerned.  We  may,  however,  draw  certain  more  or  less 
probable  inferences  from  our  knowledge  of  the  behavior  of  a  par- 
ticular part  of  the  central  nervous  system,  namely,  the  Respiratory 
Center.  In  this  region  we  have  a  rhythmic  passage  of  impulses  of  which 
the  frequency  is  determined  by  the  alternate  facilitation  and  retarda- 
tion of  conduction  which  is  brought  about,  as  we  have  seen  in  a  pre- 
ceding chapter,  by  the  presence  of  greater  or  lesser  amounts  of  Lactic 
Acid,  Carbon  Dioxide,  or  other  fatty  or  hydroxy  fatty  acids  in  the  cir- 
culating fluids.  Evidently,  therefore,  acids,  or  at  least  this  particular 
class  of  acids,  facilitate  the  passage  of  impulses  through  this  if  not 
through  other  regions  of  the  nervous  system.  Now  hyperactivity  of 
the  central  nervous  system  results  in  the  accumulation  of  acid  sub- 
stances in  the  brain,  and  we  may  with  some  probability  infer  that  the 
normal  activities  of  the  central  nervous  system  are  accompanied  to  a 
lesser  degree  by  the  production  of  similar  substances. 

THE  FATIGUE-PRODUCTS  OF  NERVE-CENTERS. 

It  has  been  pointed  out  by  Mosso  that  the  fatigue-products  of 
Nerve-centers  and  those  of  Muscle  are  probably  very  similar  in  nature 
since  mental  fatigue  is  accompanied  by  signs  of  muscular  fatigue  and 
vice  versa.  Among  the  products  of  muscular  activity  two  acids  figure 
very  largely,  namely  Lactic  Acid  and  Carbonic  Acid,  and,  if  the  products 
of  muscular  and  of  nerve-cell  activity  are  similar,  we  should  expect  to 


FATIGUE-PRODUCTS  OF  NERVE-CENTERS  525 

find  that  acids  are  set  free  in  the  central  nervous  system  as  a  result 
of  its  activity  or  fatigue.  The  actual  demonstration  of  an  increase  in 
acidity  of  the  brain-substance  as  a  result  of  prolonged  excitation  has 
proved  difficult  on  account  of  the  slightness  of  the  change  of  hydrogen 
ion  concentration  which  is  involved,  owing  to  the  buffer-action  of  the 
tissues  and  tissue-fluids,  and  the  technical  difficulties,  almost  insuper- 
able it  would  appear,  which  attend  the  utilization  of  adequate  electro- 
chemical methods  of  estimating  the  hydrogen  ion  changes  in  nervous 
tissues.  We  can,  however,  perceive  the  changed  reaction  of  the  brain 
after  excessive  stimulation  by  the  employment  of  a  simple  indicator, 
provided,  however,  that  instead  of  employing  the  change  of  color  of 
the  indicator  as  a  sign  or  measure  of  acidity,  we  employ  the  change 
in  its  solubility  in  a  solvent  which  is  immiscible  in  water. 

If  to  ten  cubic  centimeters  of  a  concentrated  (two  per  cent.)  and 
very  faintly  acid  solution  of  Neutral  Red  in  water  we  add  a  single  drop 
of  tenth-normal  potassium  hydroxide  the  color  of  the  solution  does  not 
perceptibly  change,  but  nevertheless  a  great  change  is  seen  in  respect 
to  the  lipoid-solubility  of  the  neutral  red  if  we  shake  up  the  original 
and  the  faintly  alkaline  solutions  with  Ethyl  Acetate,  from  which  any 
admixture  of  acetic  acid  has  been  previously  carefully  removed.  On 
shaking  up  with  the  faintly  acid  solution  of  neutral  red  the  ethyl 
acetate  remains  absolutely  colorless,  while  on  shaking  it  up  with  the 
faintly  alkaline  solution  the  ethyl  acetate  layer  is  stained  deep  yellow. 
In  two  ways  the  indicator  is  rendered  moire  sensitive  by  this  method; 
in  the  first  place  a  trace  of  the  yellow  modification  of  neutral  red, 
which  would  be  invisible  in  watery  solution  owing  to  the  great  excess 
of  the  red  modification,  is  removed  by  the  ethyl  acetate  and  thereby 
rendered  visible.  In  the  second  place,  let  us  suppose  that  the  Coeffi- 
cient of  Distribution: 

concentration  in  lipoid  layer 
concentration  in  aqueous  layer 

is  100  : 1  for  the  yellow  modification  of  neutral  red,  and  zero  for  the  red 
modification.  Then  at  any  given  concentration  "b"  of  hydroxyl  ions, 
if  "y"  be  the  concentration  of  the  red  modification  and  "x"  that  of  the 
yellow  modification : 

x      =  kf(b)y 

where  "k"  is  a  constant  and  f  (b)  is  some  function  of  the  alkalinity 
not  necessarily  known  or  defined.  Now  let  this  solution  be  shaken  up 
with  ethyl  acetate,  and  let  the  concentration  of  the  yellow  modi- 
fication in  the  watery  layer  now  be  "x,"  while  that  of  the  red  modifi- 
cation is  "y,"  and  that  of  the  yellow  modification  in  the  ethyl  acetate 
layer  is  "x?,"  then  we  have: 

xi      =     kf(b)yi 

x2      =      lOOxi 

x?      =      100kf(b)yi 


526     PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

that  is,  the  concentration  of  the  yellow  modification  in  the  lipoidal 
layer  (ethyl  acetate)  is  100  times  its  concentration  in  the  watery  layer 
and,  provided  f  (b)  were  a  linear  function,  it  would  be  the  same  con- 
centration as  that  which  would  be  produced  in  the  watery  layer  by  100 
times  the  concentration  of  hydroxyl  ions.  In  other  words  the  sensi- 
tiveness of  the  indicator  is  multiplied  by  the  distribution-coefficient 
of  the  lipoid-soluble  modification  between  the  two  immiscible  solvents. 
In  addition  to  this  there  is,  as  has  been  stated,  an  apparent  or  "  physio- 
logical" increase  in  the  sensitiveness  of  the  indicator  due  to  the  physical 
separation  of  the  two  colors. 

Two  frogs  may  be  taken  and  a  powerful  stimulus  applied  to  the  skin 
of  one  of  them  by  means  of  an  induced  current  for  a  prolonged  period 
(half  an  hour)  while  the  other  is  left  undisturbed.  The  brains  of  both 
animals  are  then  rapidly  removed,  divided  longitudinally  and  the  two 
parts  of  each  placed  in  a  two  per  cent,  neutral  aqueous  solution  of 
neutral  red  for  from  four  to  five  minutes.  The  two  brains  are  then 
removed  from  the  neutral  red  solution  at  exactly  the  same  moment 
and  dropped  into  neutral  ethyl  acetate. 

Within  five  or  ten  minutes  there  is  seen  to  be  a  distinct  difference 
between  the  colors  of  the  cut  surfaces  of  the  two  brains.  The  cut 
surface  of  the  brain  which  has  been  stimulated  remains  deep  red,  but 
the  indicator  diffuses  out  of  the  unstimulated  brain,  and  the  depth  of 
color  diminishes  until  it  is  only  pink.  The  differences  in  color  increase 
for  some  time,  and  in  some  instances  after  the  lapse  of  an  hour  the 
unstimulated  brain  may  be  almost  colorless,  owing  to  extraction  of  the 
dye  by  the  ethyl  acetate,  while  the  stimulated  brain  retains  a  reddish 
pink  hue.  Evidently  the  stimulated  brain  behaves  like  a  faintly  acid 
aqueous  layer,  the  unstimulated  brain  like  a  faintly  alkaline  aqueous 
layer.  The  development  of  acid  as  a  fatigue-product  of  nerve-centers 
may  thus  be  clearly  inferred. 

It  might  be  imagined  that  in  this  experiment  the  increased  acidity 
of  the  brain  may  be  apparent  and  not  real,  being  due  to  acids  carried 
to  the  brain  by  the  blood  from  the  tetanically  contracting  muscles  of 
the  stimulated  frog.  It  has  been  shown  by  Gobau,  however,  that  pre- 
cisely the  same  result  is  obtained  if  the  frog  employed  for  stimulation 
is  previously  curarized,  in  which  case  the  muscles  are  immobile. 

Acids  are  therefore  produced  in  the  brain  in  consequence  of  its 
activity  and  in  the  respiratory  center,  if  we  may  take  this  area  as  repre- 
sentative of  the  whole,  certain  specific  acids  accelerate  the  passage  of 
impulses  through  it.  We  have  thus  experimental  verification  of  the 
view  that  central  nervous  phenomena  are  self-catalyzed.  The  cata- 
•lyzer  which  is  responsible  for  the  formation  of  Memory-traces,  however, 
is  not  probably  any  substance  so  simple  as  lactic  or  carbonic  acids, 
which  as  we  have  seen,  are  stimulants  of  the  respiratory  center.  These 
substances  are  so  soluble  in  water  that  they  would  very  rapidly  be 
washed  out  of  the  nervous  tissues  and  the  persistence  of  memory- 
traces  would  be  inexplicable.  It  is  more  likely  that  we  have  here  to 


APPLICATION  OF  THE  FORMULA  OF  AUTOCATALYSIS    527 

deal  with  a  colloidal  fatty  acid  which  is  deposited  along  the  path  of 
an  impulse  and  remains  to  accelerate  or,  if  it  is  in  excess,  to  retard  a 
subsequent  impulse. 

THE  APPLICATION  OF  THE  FORMULA  OF  AUTOCATALYSIS  TO 
CENTRAL  NERVOUS  PHENOMENA. 

The  time-relations  of  any  Voluntary  Movement  are  primarily  governed 
by  events  which  occur  in  the  central  nervous  system.  This  may  readily 
be  inferred  from  the  fact  that  it  requires,  not  a  single  impulse  or  stim- 
ulus to  produce  any  coordinated  movement,  but  a  stream  of  impulses 


FIG.  46. — Photograph  of  a  drawing-board  specially  constructed  to  record  the  time- 
relations  displayed  in  the  execution  of  a  simple  volition  (the  drawing  of  a  straight 
line). 

which  must  be  maintained  throughout  the  duration  of  the  act  which 
is  performed.  A  single  stimulus,  when  applied  to  voluntary  or  striated 
muscle,  only  produces  a  single  rapid  twitch;  a  prolonged  tetanic  or 
semitetanic  movement  such  as  that  involved  in  the  performance  of 
any  muscular  exertion  is  only  possible  to  evoke  by  a  rapid  succession 
of  stimuli.  Moreover  the  performance  of  a  coordinated  muscular  act 
such  as  that  of  bending  the  arm,  involves  a  simultaneous  discharge 
of  stimulatory  impulses  to  the  flexor,  and  inhibitory  impulses  to  the 
opposing  extensor  muscles  of  the  limb. 

The  time-relations  of  a  simple  voluntary  movement,  such  as  that 
implied  in  drawing  a  straight  line  with  a  pencil  upon  a  board,  may  be 
accurately  investigated  by  a  method  which  was  originally  proposed  by 


528    PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

Loeb  and  Koranyi.  A  drawing-board  is  made  up  of  alternate  strips 
of  metal  and  wood  and  is  carefully  polished  so  that  the  junction  of  the 
strips  is  as  nearly  as  possible  indistinguishable  to  the  touch.  The 
metallic  strips  are  connected  together  in  a  circuit  which  includes  a  signal- 
magnet  and  a  metallic  pencil.  When  a  line  is  drawn  upon  the  board 
with  the  metallic  pencil  the  moment  at  which  the  pencil  touches  or 
leaves  a  metallic  strip  is  indicated  upon  a  cylinder  of  smoked  paper  by 
the  signal-magnet  (Fig.  46) .  In  this  way  the  length  of  line  traversed  at 
any  instant  in  the  entire  process  can  very  readily  be  determined.  It  is 
convenient  to  provide  a  ruler,  firmly  affixed  to  the  board,  to  guide  the 
movements  of  the  pencil  and  to  start  the  line  from  a  check  or  crotch 
in  the  ruler,  the  position  of  which  on  the  board  with  reference  to  the 
nearest  succeeding  metallic  strip  has  been  accurately  determined. 

When  the  relationship  between  the  time  and  the  extent  of  movement 
in  drawing  a  straight  line  is  investigated  in  this  way  it  is  found  that  the 
Autocatalytic  Formula: 

log  — — —      =     ka(t   -  ti) 
a    —  x 

applies  with  remarkable  accuracy,  "a"  being  the  total  length  of  the 
line,  "x"  the  length  of  line  drawn  at  time  "t"  after  the  motion  of  the 
pencil  first  began,  and  "t,"  the  time  taken  to  reach  the  middle  of  the 
line.  The  following  is  an  illustrative  result: 

Subject  R.M.M.rf  Formula:     log  — — -^ —         =     k  (t   -  38.75) 

ol .  0    —  x 

.    x  t  k 

inches.  1/100  sees. 

1 10.00  0.051 

2 15.50  0.050 

3 17.50  0.046 

4 21.00  0.047 

5  ............  22.50  0.044 

6 ,      ...  25.00  0.045 

7  .......  26.00  0.042 

8  .      ,     .      .      .      ...      .      .      .      .     27.50  0.041 

9 29.00  0.040 

10 30.75  0.040 

11 32.00  0.038 

12 33.50  0.038 

13 35.00  0.038 

14  ,     .     i 36.50  0.037 

15 38.00  0.037 

16 39.50  0.037 

17 40.75  0.042 

18 42.00  0.043 

19 43.50  0.042 

20  .      ...      .      .      .•    .      .      .      ...      .  45.00  0.041 

.............  47.00          .  0.039 

22  •      •      .....      .      .      .      ...  48.25  0.041 

23  •      •      v    •      *      .      .      .      .      .      .      .  50.00  0.041 

24 .      -:.'.'.'.      .      .  52.00  0.044 

25  •      •      •     V    '.      ."'     .      .      ...      .      .  53.75  0.041 

26 :..  55.50  0.043 

27 57.50  0.044 

28 59.50  0.049 

29 61.75  0.050 


30 


66.00  0.054 


APPLICATION  OF  THE  FORMULA  OF  AUTOCATALYSIS    529 

The  values  in  the  third  column  are,  as  would  be  required  by  the 
formula  of  autocatalysis,  almost  constant.  The  performance  of  this 
particular  type  of  central  nervous  activity  is  therefore  autocatalyzed. 

Turning  now  to  the  much  more  complex  phenomenon  of  Memory 
we  are  in  possession  of  quantitative  data  which  have  been  most  elabor- 
ately compiled  by  the  psychologist  Ebbinghaus.  The  method  which 
he  employed  was  to  read  and  reread  a  series  of  meaningless  syllables 
at  a  definite  rate,  0.40  seconds  being  expended  in  the  perusal  of  each 
syllable.  The  data  recorded  are  the  numbers  of  repetitions  which  were 
found  to  be  necessary  to  attain  the  perfect  memorization  of  the  given 
number  of  syllables  in  the  series.  Hence  the  time  in  seconds  which  was 
employed  in  learning  each  series  was  0.4  X  n  X  r  where  "n"  and  "r" 
were  the  number  of  syllables  in  the  series  and  the  number  of  repetitions 
respectively.  Excepting  in  the  case  of  the  first  observation  (that  is, 
the  number  of  syllables  learnt  in  a  single  repetition)  the  syllables  were 
read  in  conjunction  with  a  sufficient  quantity  of  other  material  to 
make  the  total  length  of  each  period  of  reading  approximately  the 
same.  The  following  were  the  results  obtained: 

Number  of  syllables.  Number  of  repetitions.  Time  in  seconds. 
7                                                         1.0  2.8 

12  16.6  79.7 

16  30.0  192.0 

24  44.0  422.4 

36  55.0  792.0 

If  we  apply  to  these  results  the  formula  of  autocatalysis,  calling 
"a"  the  maximal  number  of  syllables  which  Ebbinghaus  could  have 
memorized  by  any  number  of  repetitions,  "x"  the  number  actually 
learnt  or  the  extent  of  the  trace  or  deposit  formed  in  time  "t,"  and 
"t/'  the  time  consumed  in  learning  half  the  maximal  number,  we  find 
that  the  following  equation  most  nearly  expresses  the  results: 


0.001468     t    -  0.526 


In  the  following  table  the  experimental  values  of  "x"  and  those 
calculated  from  the  formula  are  compared  : 

Time  in  seconds  x  (observed)  x  (calculated). 

2.8     ....      .......       7  10.1 

79.7     .......      .....      12  12.2 

192.0     .........      .      .      16  15.8 

422.4     ...........      24  24.2 

792.0     ...........     36  35.4 

The  only  deviation  of  significant  magnitude  is  that  between  the 
observed  and  calculated  numbers  of  syllables  which  may  be  learnt  in 
a  single  repetition.  This,  however,  may  most  probably  be  attributed 
to  the  conditions  under  which  this  number  was  determined,  differing 
as  they  did,  by  the  non-inclusion  of  other  reading  matter,  from  the 
conditions  which  pertained  in  the  remaining  observations. 
34 


530    PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

The  view  that  the  formation  of  the  Memory-trace  is  due  to  an  auto- 
catalyzed  chemical  reaction,  therefore,  not  only  enables  us  to  interpret 
some  of  the  most  striking  qualitative  phenomena  of  intellectual  proc- 
esses, but  also  to  predict  their  quantitative  alteration  with  successive 
repetition.  The  quantitative  data  obtained  by  Ebbinghaus  are  among 
the  most  readily  interpretable  and  at  the  same  time  accurate  measure- 
ments of  this  kind  which  we  possess,  but  a  variety  of  measurements 
which  have  been  made  on  the  rate  of  learning  by  telegraph-operators, 
typists,  and  so  forth,  all  yield  "  curves  of  learning"  which  very  strikingly 
resemble  the  curve  which  represents  the  progress  of  an  autocatalyzed 
chemical  reaction,  and  in  some  cases,  it  appears,  two  or  more  of  such 
curves  may  be  superimposed  to  yield  "cycles  of  learning"  just  as  we 
have  cycles  of  growth  in  a  growing  organism. 


SLEEP. 

The  various  theories  of  sleep  which  have  been  proposed  are  no  less 
numerous  than  those  which  have  been  propounded  to  account  for 
the  phenomenon  of  memory.  A  Vasomotor  Theory  of  Sleep  has  been 
advanced  by  Howell,  who  considers  that  it  is  attributable  to  cerebral 
anemia,  due  to  a  diminished  blood-supply  to  the  brain,  following  the 
general  fall  of  arterial  pressure  which  accompanies  sleep.  While  this 
may  very  possibly  be  a  contributing  factor  to  the  phenomenon  of  sleep, 
yet,  on  the  other  hand,  it  is  at  least  equally  conceivable  that  the  vaso- 
motor-phenomena  which  accompany  sleep  are  merely  secondary  mani- 
festations of  the  processes  which  induce  sleep,  and  that  the  actual 
onset  of  sleep  is  due  primarily  to  other  factors.  The  close  connection 
of  sleep  with  Fatigue  on  the  one  hand,  and  with  the  absence  or  monot- 
ony of  Sensory  Stimulation  on  the  other,  indicates  very  clearly  that  a 
condition  of  the  nervous  tissues  consequent  upon  prolonged  activity 
is  a  potent  factor  predisposing  the  central  nervous  system  to  the 
relatively  suspended  activity  of  sleep. 

The  accumulation  of  Fatigue  Products  in  the  brain,  when  it  has 
exceeded  the  amount  which  causes  maximal  facilitation  of  the  passage 
of  nervous  impulses,  begins  to  retard  the  passage  of  impulses,  and  this 
retardation  increases  with  the  degree  of  accumulation.  With  continued 
wakefulness,  as  many  observers  have  pointed  out,  the  Threshold  of 
Sensory  Stimulation  rises.  A  stronger  stimulus  than  usual  is  required 
to  traverse  the  clogged  and  overloaded  channels,  and  consequently  the 
environment,  by  exclusion  of  the  countless  slight  fluctuating  impres- 
sions which  lend  variety  to  our  surroundings,  becomes  more  and  more 
monotonous,  fewer  and  fewer  "channels"  of  the  brain  are  traversed 
by  impulses,  larger  and  larger  areas  become  quiescent  through  lack 
of  traversing  stimuli,  until  finally  sleep  supervenes,  and  the  whole  of 
the  brain  except  those  portions,  chiefly  in  the  medulla,  which  are 
vital  to  the  maintenance  of  the  circulation  and  respiration,  and  some 


SLEEP  531 

detached  fragment  which  may  be  occupied  in  weaving  dreams,  has 
subsided  into  quiescence. 

It  is  the  variety  of  our  environment  and  the  intensity  of  rapidly 
succeeding  sensory  impulses  which  keep  us  awake,  by  forming  new 
"channels"  which  intersect  with  other  channel-systems,  i.  e.,  arouse 
"associations"  and  keep  up  a  continuous  activity  over  the  whole  area 
of  consciousness.  If  the  Field  of  Consciousness  is  limited,  either  by 
fatigue  or  by  the  limitation  of  incoming  sensory  impressions,  one 
group  after  another  of  channel-systems  or  interconnected  memory- 
traces  sink  into  quiescence  until  only  the  least  fatigued  or  the  most 
intensely  stimulated  channels  are  awake.  When  the  stimulation  is 
nowhere  sufficient  to  rise  above  the  threshold  of  consciousness,  we 
have  sleep,  but  where  the  stimulation  is  intense,  and  yet  excessively 
circumscribed,  we  have  the  condition  of  Hypnosis.  The  extraordinary 
vividness  of  the  impressions  which  are  formed  under  hypnosis  is  due 
to  the  isolation  of  these  impressions  and  to  the  fact  that  for  the  moment 
the  brain  is,  for  all  effective  purposes,  limited  to  and  circumscribed  by 
the  areas  which  are  directly  stimulated.1  Inhibitive  and  conflicting 
impressions  are  temporarily  in  abeyance. 

The  customary  method  by  which  we  recollect  past  events  is  the 
Association  of  a  present  event  with  an  incident  which  recalls  the  past. 
In  other  words  a  stimulus  of  the  present  moment  happens  to  traverse 
a  previously  formed  system  of  trace-deposits.  If,  however,  only  a  small 
portion  of  the  brain  be  active  the  chance  of  a  subsequent  impulse 
traversing  it  must  obviously  be  less  than  when  the  area  of  stimulation 
is  larger.  The  cutting  off  of  sensory  impressions  in  sleep  and  the 
diminution  of  the  extent  and  variety  of  "canalization"  or  trace-for- 
mation throughout  the  upper  portion  of  the  central  nervous  system 
which  accompanies  sleep  is  therefore  conducive  to  Amnesia  or  lack  of 
ability  to  recollect  the  intellectual  events  which  occur  under  these 
circumstances.  This  fact  is  well  illustrated  by  phenomena  which  fre- 
quently attend  the  onset  of  sleep.  A  certain  sequence  of  ideas  arises  in 
the  consciousness — we  think  of  it,  as  we  say,  dreamily — then  suddenly 
this  train  of  ideas  vanishes  and  another  takes  its  place,  and  we  find 
that  we  cannot  recollect  the  first.  This  amnesia  is  occasionally  so 
surprising  in  itself  that  the  wonder  of  it  excites  us  to  the  extent  of 
awakening.  So  the  cessation  of  canalization  in  one  trace-system 
leads,  by  the  blocking  off  of  impulses,  to  its  cessation  in  an  adjacent 
system,  and  amnesia  spreads  over  a  wider  and  wider  area,  until  finally 
sleep  supervenes.  The  fabric  of  intercommunicating  trace-systems 
which  constitutes  the  waking  consciousness  shows  larger  and  larger 
rents  of  amnesia,  the  fragments  of  the  fabric  are  less  and  less  bound 
together,  until  at  last  the  entire  fabric  seems  to  be  blotted  out,  or  one 

1  The  impressions  received  during  hypnosis  are  usually  separated  from  the  waking 
impressions  by  a  gap  of  amnesia,  but  during  the  actual  period  of  hypnosis  the  extra- 
ordinary vividness  of  the  impressions  received  is  testified  by  the  almost  automatic 
response  of  the  body  to  commands  or  suggestions  which  are  received  in  this  condition. 


532    PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

shred  may  remain,  as  in  a  dream,  to  be  faintly  recalled  or  completely 
forgotten  in  awakening,  according  to  whether  or  not  our  customary 
waking  perceptions  (traces)  traverse  the  point  of  union  of  the  dream- 
shred  with  the  whole  fabric  of  the  reawakened  consciousness. 

That  the  onset  of  sleep  is  in  reality  due  to  the  accumulation  of 
Fatigue-products  which  are  washed  out  during  the  period  of  quiescence 
by  the  circulatory  fluids,  has  been  very  strikingly  demonstrated  by 
Pieron.  This  observer  has  shown  that  if  the  blood-serum  or  Cerebro- 
spinal  Fluid  of  a  dog  which  has  been  kept  awake  for  an  abnormal 
period  be  injected  directly  into  the  fourth  ventricle  of  the  brain  of  a 
normal  dog,  even  if  this  latter  animal  has  recently  slept,  it  falls  at 
once  into  a  profound  slumber.  The  effect  is  much  greater  if  cerebro- 
spinal  fluid  or  the  fluid  from  the  ventricles  of  the  brain  is  employed  than 
if  blood-serum  be  used.  Frequently  with  blood-serum  nothing  more 
than  a  moderate  somnolence  is  elicited,  whereas  when  cerebrospinal 
fluid  is  employed  the  slumber  which  is  induced  may  be  so  profound 
that  the  animal  will  remain  asleep  in  any  attitude  in  which  he  may  be 
placed.  Pieron  has  made  many  interesting  observations  upon  the 
chemical  nature  of  this  sleep-inducing  substance,  or  Hypnotoxin  as 
he  designates  it.  He  finds  that  it  is  destroyed  by  heating  to  65°  C.  and 
by  oxidation,  is  precipitable  or  coagulable  by  alcohol  and  is  non- 
diffusible.  It  is  evidently,  therefore,  a  colloidal  substance  of  some 
complexity,  and  chemically  unstable. 

THE  FADING  OF  MEMORY-TRACES. 

It  is  a  matter  of  common  experience,  and  a  fact  which  has  been 
experimentally  verified,  that  a  person  who  has  been  deprived  of  sleep 
beyond  the  normal  period  of  wakefulness  does  not  require  the  full  sum 
of  the  periods  of  sleep  which  he  has  lost  in  order  completely  to  recover 
from  his  desire  to  sleep.  We  must  therefore  conclude  that  not  only  do 
Fatigue-products  disappear  from  the  brain  during  sleep  but,  furthermore, 
that  they  disappear  the  more  rapidly  the  greater  their  concentration. 
We  have  seen  that  the  initial  effect  of  the  fatigue-products  of  the  cen- 
tral nervous  tissues  is  to  cause  facilitation  of  the  passage  of  nervous 
impulses  and  the  formation  of  Memory-traces.  The  phenomenon  of 
forgetting  must  therefore  be  essentially  of  the  same  nature  as  the  phe- 
nomenon of  refreshment  by  sleep,  i.  e.,  it  must  consist  in  (or  depend 
upon)  the  disappearance  of  the  products  of  their  functional  activity 
from  certain  nerve-tracts. 

Ebbinghaus  has  carried  out  a  number  of  excessively  painstaking 
investigations  upon  the  rate  at  which  meaningless  syllables  which  have 
once  been  learned  by  heart  are  forgotten.  Ebbinghaus  was  his  own 
subject.  Series,  each  consisting  of  thirteen  meaningless  syllables,  were 
read  and  reread  in  such  a  manner  that  each  syllable  was  presented 
to  the  senses  for  a  period  of  0.41  seconds  at  each  repetition.  When 
it  was  found  just  possible  to  completely  recall  the  series  correctly, 
the  total  time  (=  ti)  consumed  in  memorizing  the  series  was  noted. 


FADING  OF  MEMORY-TRACES  533 

After  the  lapse  of  certain  definite  periods  of  time  the  series  were 
relearned,  and  the  time  (=  t]  --  t)  necessary  to  relearn  them  was 
also  noted.  Then  the  difference  (=  t)  represented  the  time  saved  by 
the  previous  repetitions,  or  in  other  words  the  time  which  would  be 
consumed  in  learning  that  proportion  of  syllables  which  was  remem- 
bered. The  percentage  -  X  100  was  employed  by  Ebbinghaus  (and 

ti 

has  been  employed  by  his  successors  in  this  field  of  investigation)  as 
the  most  convenient  measure  of  the  extent  of  forgetting.  It  is,  of 
course,  not  actually  equivalent  to  the  amount  of  memorized  material 
which  has  been  forgotten,  for  the  time  required  to  memorize  syllables 
is,  as  we  have  seen,  not  proportional  to  their  number.  Nevertheless 
the  outline  of  the  relationship  between  the  time  which  has  elapsed 
since  the  material  was  learned  and  the  amount  of  material  forgotten 

is  sufficiently  clearly  revealed  by  the  successive  values  of  -     -  X  100 

ti 

noted  by  Ebbinghaus  to  show  that  this  phenomenon,  like  that  of 
refreshment  by  sleep,  occurs  most  rapidly  in  the  beginning,  when  the 
mass  of  deposit  undergoing  destruction  or  dispersal  is  greatest.  The 
following  were  the  results  obtained  by  Ebbinghaus — the  time  being 
reckoned  from  the  end  of  the  first  period  of  learning  to  the  end  of  the 
second . 


Time  in  hours.  -    X   100 

ti 

0.33 41.8 

1.00 .      .  55.8 

8.80     .      .      .      . .      .  ....  64.2 

24.00 66.3 

48.00 72.2 

144.00 74.6 

744.00 78.9 

The  negative  acceleration  of  this  process  is  extraordinarily  high,  for 
although  over  55  per  cent,  of  the  time  saved  by  the  first  period  of 
learning  is  lost  in  one  hour,  yet  during  the  succeeding  twenty-three 
hours  only  9  per  cent,  more  is  lost.  In  other  words  the  Velocity  of 
Forgetting  decreases  very  rapidly  with  the  passage  of  time;  it  never, 
under  normal  conditions,  undergoes  any  increase  in  rapidity  with  time. 
The  process  of  forgetting  is  therefore  essentially  different  in  mechanism 
from  the  process  of  memory-formation. 

It  is  very  improbable  that  the  fading  of  a  memory-trace  can  be  due 
to  chemical  changes  in  the  substance  forming  the  trace,  for  no  chemical 
reactions  are  known  which  diminish  so  greatly  in  rapidity  with  time, 
continue  to  proceed,  and  yet  do  not  attain  completion  for  such  pro- 
longed periods  as  the  memory-traces  persist.  A  reaction  which  was  55 
per  cent,  completed  in  one  hour  would  either  have  ceased  before 
twenty-four  hours,  or  else  would  be  much  more  than  66  per  cent,  com- 
pleted. A  chemical  reaction,  to  display  such  extraordinary  falling-off 


534    PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

in  velocity  with  time,  would  have  to  be  polymolecular,  i.  e.,  involve  a 
large  number  of  simultaneously  reacting  molecules,  and  polymolecular 
reactions  do  not  actually  occur,  or  rather  they  take  place  in  successive 
monomolecular,  bimolecular  or  trimolecular  stages. 


Tim  e  in  6-46  hou  r  units 


FIG.  47. — Curves  illustrating  the  analogies  between  the  fading  of  a  memory  trace, 
the  extraction  of  protamine  from  spermatozoa  by  acid  and  the  dissolution  of  dried 
casein  by  dilute  alkali. 

If,  however,  we  compare  the  curve  of  forgetting  with  the  curve  which 
expresses  the  rate  of  issuance  of  a  colloid  (or  possibly  of  a  crystalloid) 
from  a  colloidal  into  a  fluid  menstruum,  we  cannot  fail  to  recognize 


FADING  OF  MEMORY-TRACES  535 

at  once  their  essential  similarity.  In  the  accompanying  figure  (Fig. 
47)  curve  1  represents  the  rate  of  issuance  of  potassium  caseinate 
from  suspended  Casein  particles  into  dilute  potassium  hydroxide  solu- 
tion; 2  represents  the  rate  of  extraction  of  Protamine  (salmin)  from 
dried  salmon-spermatozoa  by  dilute  hydrochloric  acid;  and  3  represents 
the  Curve  of  Forgetting,  as  illustrated  by  the  results  of  Ebbinghaus 
cited  above.  Comparing  these  curves  it  is  evident  that  by  a  suitable 
modification  of  parameters  any  one  of  them  might  be  employed  in 
place  of  the  others  to  illustrate  the  processes  which  they  severally 
depict,  and  that  each  of  them  represents  the  time-relations  of  a  process 
in  which  the  negative  acceleration  is  so  marked  as  to  forbid  its  repre- 
sentation by  any  known  chemical  reaction-formula,  or  by  the  similar 
formulae  which  represent  the  diffusion  of  crystalloids  in  fluid  media. 
It  has  been  found  that  the  issuance  of  a  protein  (and  therefore,  probably 
of  other  colloids)  from  a  colloidal  menstruum  is  governed  primarily 
by  Capillary  Forces  so  that  the  time-relations  of  the  washing-out 
process  are  similar  to  those  exhibited  in  the  rise  of  a  fluid  in  a  capil- 
lary tube  or  of  a  liquid  in  a  column  of  sand  or  a  strip  of  filter-paper. 
We  may  infer  that  the  fading  of  a  memory-trace  is  attributable  to 
some  similar  phenomenon  and  may  not  improbably  be  due  to  the  wash- 
ing out  of  a  colloidal  substance,  which  forms  the  memory-trace,  by 
the  circulating  fluids.  This  would  explain  at  once  the  rapidity  of  the 
initial  stages  of  forgetting  and  the  extraordinary  persistence  of  the 
last  traces  of  the  memory-deposit,  for  complete  extraction  of  a  colloid 
from  a  colloidal  menstruum  by  an  external  liquid  is  a  matter,  not  of 
hours,  but,  as  may  be  computed  by  exterpolation  from  actual  measure- 
ments, may  actually  require  the  lapse  of  periods  of  time  which  are 
vastly  in  excess  of  the  total  duration  of  the  life  of  man. 


REFERENCES. 
MEMORY: 

Maudsley:     Body  and  Mind,  London,  1873.     The  Pathology  of  Mind,  London,  1879. 

Munk:     Ueber  die  Funktionen  der  Gehirnrinde,  Berlin,  1881. 

Exner:     Pfliigei's   Arch.,    1882,    28,    p.    487.     Entwurf    zu    einer   physiologischen 

Erklarung  der  psychischen  Erscheinungen,  Wien,  1894. 
Ebbinghaus:     Ueber  das  Gedachtniss,  Leipzig,  1885. 
Loeb  and  Koranyi:     Pfliiger's  Arch.,  1890,  46,  p.  101. 
Mosso:     Arch.  f.  Anat.  u.  Physiol.,  Physiol.  Abt.,  1890,  p.  129.     Phil.  Trans.  Roy. 

Soc.,  London,  1892.  183,  p.  299. 
Lepine:     Revue  de  Medicine,  1894,  p.  727. 
Duval:     Compt.  rend,  de  la  Soc.  Biol.,  1895,  p.  85. 
Smith:     Psychological  Review,  1896,  3,  p.  21. 
Bryan  and  Harter:     Ibid.,  1897,  4,  p.  27;  1899,  6,  p.  345. 
Loeb:     Comparative  Physiology  of  the  Brain  and  Comparative  Psychology,  New 

York,    190C. 

James:     Principles  of  Psychology,  London,  1901. 
Robertson:     Arch;  Internat.  de  Physiol.,  1908,  6,  p.  388.     Biochem.  Zeit.  Festband. 

f.  H.  J.  Hamburger,  1908,  p.  287.      Folia  Neurobiologica,  1912,  6,  p.  553;  1913. 

7,  p.  309. 

Gobau:     Ann.  et  Bull,  de  la  Soc.  de  Med.  de  Gand.,  1910,  76,  No.  4. 
Pieron:     L'Annee  Psychologique,  1913, 19,  p.  91.     Principles  of  Psychology,  London, 
1901. 


536    PROCESSES  INFERRED  FROM  INDIRECT  OBSERVATION 

HYPNOSIS: 

Bernheim:     Suggestive  Therapeutics,  trans,  by  Herter,  New  York,  1888. 

Moll:     Hypnotism,  London,  1890. 

Sidis:     The  Psychology  of  Suggestion,  New  York,  1911. 

Prince:     The  Unconscious,  New  York,  1915. 

Ochorwicz:     Hypnotisme,  Richet's  Dictionnaire  de  Physiologie,  Paris. 
SLEEP: 

Sidis:    An  Experimental  Study  of  Sleep,  Boston,  1909. 

Manaceine:     Sleep,  its  Physiology,  Pathology,  Hygiene  and  Psychology,  London, 
1912. 

Pier  on:     Le  Pro bl erne  Physiologique  du  Sommeil,  Paris,  1913. 
FORGETTING: 

Ebbinghaus:     Vide  supra. 

Patrick  and  Gilbert:     Psychological  Review,  1896,  3,  p.  469. 

Swift:     Psychological  Bulletin,  1906,  3,  p.  185;  1910,  7,  p.  17. 

Bean:     Columbia  Contributions  to  Philosophy  and  Psychology,  1912,  20,  No.  3. 

Robertson:     Folia  Neurobiologica,   1914,  8,  p.  485. 
RATE  OF  EXTRACTION  OF  COLLOIDS  FROM  COLLOIDAL  MENSTRUA: 

Cameron  and  Bell:     U.  S.  Dept.  Agr.,  Bureau  of  Soils,  Bull.  No.  30.     Jour.  Physical 
Chem.,  1906,  10,  p.  658. 

Ostwald:     Zeit.  f.  Chem.  u,  Ind.  der  Kolloide,  2d  Supplement,  1908,  2,  p.  xx. 

Robertson:     Jour.  Phys.  Chem.,  19^0,  14,  p.  377.     Jour.  Biol.  Chem.,  1913    14   p. 
237.     Pfluger's  Arch.,  1913,  152,  p.  524. 

Robertson  and  Miyake:     Jour.  Biol.  Chem.,  1916,  25,  p.  351;  1916,  26,  p.  129. 


PART  V. 

THE  PRODUCTS  OF  TISSUE-ACTIVITY 


CHAPTER  XXII. 
THE  WASTE-PRODUCTS. 

THE  CARBONACEOUS  WASTE-PRODUCTS. 

The  chief  carbonaceous  waste-product  is,  of  course,  Carbon  Dioxide. 
Only  a  trifling  proportion  of  the  excretion  cf  carbon  dioxide  takes 
place  through  the  urine,  feces  and  sweat,  the  lungs  playing  the  pre- 
ponderating part  in  accomplishing  the  elimination  of  this  product. 
The  total  production  of  carbon  dioxide  in  twenty-four  hours  varies 
with  the  quality  and  quantity  of  food  ingested,  with  the  quantity  of 
muscular  work  performed,  and  with  the  rate  or  loss  of  heat  from  the 
body,  but  in  an  adult  male  doing  moderate  work  it  may  be  estimated 
in  round  numbers  at  four  hundred  liters  at  ordinary  temperatures  and 
atmospheric  pressure. 

The  carbon-dioxide  output  is  derived  from  the  oxidation  of  the 
carbon  in  the  metabolized  foodstuffs.  It  arises,  therefore,  in  conse- 
quence of  the  absorption  of  oxygen  by  the  tissues.  Carbon  dioxide  is, 
however,  not  the  only  oxidation-product  of  cellular  activities,  and 
hence  the  carbon  dioxide  which  is  given  off  by  an  animal  is  rarely  the 
molecular  equivalent  of  the  oxygen  which  is  absorbed  in  the  same 

period.    The  ratio:  -  ,     is  termed  the  Respiratory  Quotient 

O2  absorbed 

and  it  varies  in  a  very  characteristic  manner  with  the  nature  of  the 
ingested  foodstuffs.  Thus  the  carbohydrates  contain  a -greater  pro- 
portion of  oxygen  than  any  of  the  other  foodstuffs,  the  oxygen  being, 
in  fact,  molecularly  equivalent  to  the  hydrogen  which  they  contain. 
The  hydrogen  in  a  carbohydrate  may,  therefore,  be  regarded  as  having 
been  completely  oxidized  beforehand,  and  the  carbohydrates  behave, 
so  far  as  the  absorption  of  oxygen  and  evolution  of  carbon  dioxide  are 
concerned,  as  if  they  consisted  of  pure  carbon  and  underwent  the 
reaction : 

c  +  O2  =  co2 


53$  WASTE-PRODUCTS 

hence  the  respiratory  quotient  for  the  oxidation  of  pure  carbohydrates 
is  equal  to  unity.  This  probably  represents  the  maximal  value  of  the 
respiratory  coefficient  which  may  be  obtained  with  normal  animal  tis- 
sues. Figures  in  excess  of  this  which  have  occasionally  been  observed 
have  been  attributed  by  some  observers  to  the  formation  in  the  tissues 
of  fat  from  carbohdrates  with  the  liberation  of  carbon  dioxide: 


+     O2     =     Ci6H32O2     +     8CO2     +     8H2O 
Glucose.  Palmitic  acid. 

Respiratory  quotients  in  excess  of  unity  have  been  observed  in  hiber- 
nating animals  immediately  prior  to  their  winter-sleep,  and  in  animals 
and  birds  fed  with  an  enormous  excess  of  carbohydrates. 

The  respiratory  quotient  for  the  oxidation  of  Fats  is  necessarily 
much  lower  than  it  is  for  carbohydrates,  since  the  fats  do  not  contain 
more  than  about  one-sixth  of  the  oxygen  which  is  required  to  convert 
the  hydrogen  which  they  contain  into  water.  An  important  proportion 
of  the  absorbed  oxygen  is  therefore  excreted  in  the  form  of  water,  and 
the  carbon  dioxide  which  is  discharged  from  the  body  falls  very  much 
short  of  the  molecular  equivalent  of  the  oxygen  absorbed,  the  respira- 
tory quotient  for  the  ordinary  dietary  fats  being  0.71.  The  Proteins 
contain  about  half  the  oxygen  needed  to  oxidize  their  hydrogen  and  the 
respiratory  quotient  is  intermediate  between  the  value  for  carbohydrate 
and  fats,  namely  0.81.  'The  respiratory  quotient  for  Alcohol  is  lower 
even  than  for  fats,  namely  0.67. 

From  these  considerations  it  is  evident  that  the  value  of  the  respira- 
tory coefficient  must  be  capable  of  yielding  important  information  as 
to  the  particular  class  of  foodstuffs  which  is  being  utilized  for  the 
performance  of  a  given  function.  Thus  for  man,  under  ordinary  con- 
ditions of  work  and  nourishment,  the  respiratory  quotient  lies  between 
0.8  and  0.9,  but  when  hard  Muscular  Work  is  being  performed  it  rises 
and  may  even  approach  the  ideal  value  of  unity  for  the  oxidation  of 
carbohydrates.  Part  of  this  rise,  especially  during  the  initial  stages 
of  a  work-experiment,  or  in  experiments  occupying  only  a  short  period, 
may  possibly  be  ascribed  to  the  "washing  out"  of  carbon  dioxide 
accumulations  from  the  tissues  by  the  more  rapid  respiratory  and 
cardiac  movements.  It  must  be  recollected,  however,  that  the  rapidity 
of  the  respiratory  movements  in  exercise  is  conditioned  by  the  enhance- 
ment of  the  carbon-dioxide  content  of  the  blood,  so  that  but  a  slight 
proportion  of  the  increased  carbon-dioxide  output  during  exercise  can 
justifiably  be  attributed  to  the  increased  ventilation  of  the  body,  and, 
furthermore,  the  effect  of  muscular  work  upon  the  respiratory  quotient 
endures  for  a  long  period,  until  in  fact,  the  carbohydrate-reserves  have 
been  so  far  depleted  that  we  may  surmise  that  other  foodstuffs  are 
now  being  utilized  for  the  production  of  muscular  energy.  The  rise 
of  the  respiratory  quotient  during  the  performance  of  muscular  work 
therefore  affords  us  confirmatory  evidence  of  the  view  that  muscular 


CARBONACEOUS  WASTE-PRODUCTS  539 

energy  is  derived,  in  the  first  place,  from  the  oxidation  of  carbo- 
hydrates. 

On  the  other  hand,  in  Starvation,  the  respiratory  quotient  falls  to  a 
value  intermediate  between  that  characteristic  for  the  oxidation  of 
fats  and  the  value  for  proteins,  for  in  starvation  the  carbohydrate 
reserves  are  quickly  depleted,  and  thereafter  the  energy  which  is  dis- 
sipated by  the  body  is  derived  from  the  oxidation  of  the  fat  reserves 
and  the  tissue  proteins. 

Extraordinarily  low  values  of  the  respiratory  quotient  have  occa- 
sionally been  obtained  with  Hibernating  Animals  during  their  winter- 
sleep.  Thus  Pembrey  obtained  figures  as  low  as  0.25  with  hibernat- 
ing dormice.  For  hibernating  bats  Hari  obtained  higher  figures,  but 
even  these  values  were  generally  less  than  the  normal  value  for  the 
oxidation  of  pure  fats.  The  origin  of  these  low  values  has  been  the 
subject  of  numerous  surmises.  It  appears  to  be  incontestable  that 
they  represent  incomplete  oxidations,  which  do  not  proceed  so  far  as 
to  result  in  the  formation  of  carbon  dioxide.  A  question  much  more 
difficult  to  decide,  however,  is  whether  the  excess  of  oxygen  intake 
over  carbon-dioxide  output  in  the  winter-sleep  is  stored  in  the  animal's 
tissues,  or  excreted  in  the  form  of  compounds  other  than  carbon 
dioxide.  It  was  at  first  supposed  that  the  oxygen  excess  was  stored  in 
the  tissues  in  the  form  of  partially  oxidized  foodstuffs,  as,  for  example, 
carbohydrates  derived  from  fats.  It  has  been  pointed  out,  however, 
that  the  total  accumulation  of  oxygen  throughout  the  duration  of  the 
winter-sleep  would  necessitate  the  production  of  a  quantity  of  carbo- 
hydrate far  in  excess  of  the  total  carbohydrate-content  of  the  animals 
under  any  conditions.  It  has  been  ascertained  that  the  urine  of 
hibernating  animals  contains  notable  quantities  of  products  of  incom- 
plete oxidation  such  as  Lactic  Acid,  and  it  is  probable  that  a  con- 
siderable proportion  of  the  excess  of  absorbed  oxygen  is  excreted  in 
the  urine  in  these  forms. 

Not  only  does  the  ratio  of  carbon-dioxide  evolved,  to  oxygen  ab- 
sorbed, rise  during  the  performance  of  muscular  exercise,  but  the 
total  carbon-dioxide  output  increases  in  direct  proportion  to  the  work 
performed.  This  has  been  shown  in  a  very  striking  manner  by  the 
experiments  of  Johansson  who  first  measured  his  carbon-dioxide  out- 
put per  hour  at  rest  and  then  during  the  performance  of  the  Muscular 
Work  involved  in  repeatedly  lifting  a  weight.  He  found  that  his  carbon- 
dioxide  output  rose  to  the  value 

CO2    =    Np   +    q 

where  "  q"  was  the  output  at  rest,  "N"  the  number  of  times  the  weight 
was  lifted  and  "p"  the  increase  in  output  induced  by  lifting  the  weight 
once. 

The  effect  of  the  Temperature  of  the  environment  upon  the  carbon 
dioxide  output  is  opposite  in  cold-blooded  and  warm-blooded  animals. 


540  WASTE-PRODUCTS 

In  cold-blooded  animals,  in  which  the  temperature  of  the  tissues  approx- 
imates to  that  of  the  environment,  the  rate  of  oxidations  is  increased, 
as  might  be  expected,  by  a  rise  in  external  temperature,  and  the 
carbon-dioxide  output  is  even  more  than  proportionately  increased, 
since  the  respiratory  quotient  generally  undergoes  a  slight  rise  with 
temperature  also.  This  is  illustrated  by  the  following  experiments  of 
C.  J.  Martin  on  the  carbon  dioxide  output  of  the  Australian  Lizard 
Cyclodus  gigas. 

COz  output  per  kilogram 

Temperature  of  Temperature  of  and  hour, 

the  air.  the  animal.  mg. 

5 .      .  5.5  13 

9 9.2  42 

15 15.2  53 

20.5 20.4  55 

25.        .. 24.5  64 

30.        ....      .      .      .      .    V     .      .      .  29.3  78 

35.       -.-•;• v     .      .      .  34.8  97 

39. 38.5  292 

The  effect  of  rising  temperature  upon  the  carbon-dioxide  output  of 
warm-blooded  animals  is,  within  certain  limits,  the  reverse  of  this. 
The  Body-temperature  of  the  warm-blooded  animals  varies  but  slightly 
with  the  temperature  of  the  environment  and  this  uniformity  of  tem- 
perature is  secured  by  a  number  of  cooperating  factors,  among  which 
the  radiation  of  heat  from  the  surface  of  the  body,  the  loss  of  heat  by 
the  latent  heat  of  evaporation  of  perspiration,  and  the  adjustment  of 
the  production  of  heat  by  the  oxidations  of  the  body  to  the  need  for 
heat  to  maintain  the  normal  temperature  of  the  tissues.  The  increase 
of  metabolism  which  low  temperatures  induce  in  the  warm-blooded 
animals  is  probably  brought  about,  in  part  at  least,  by  the  stimulation 
of  the  skin  by  cold  air  inducing  reflex  movements,  such  as  shivering 
or  reflex  alterations  of  muscular  tone  which  necessitate  an  enhanced 
combustion  of  carbohydrates  with  the  performance  of  a  minimum  of 
external  work. 

The  regulation  of  the  temperature  of  the  body  between  the  normal 
"comfortable"  temperature-limits  of  the  environment  is  mainly 
brought  about  by  the  modification  of  the  purely  physical  factors  of 
radiation  and  evaporation  which  govern  the  rate  of  loss  of  heat  from 
the  body.  Below  the  external  temperature  of  20°  C.  (68°  R),  however, 
the  "chemical  regulation"  of  the  bodily  temperature  becomes  an 
exceedingly  important  factor,  the  rate  of  metabolism  rising  continu- 
ously, and^considerably  with  falling  temperature.  Above  30°  C.-350  C. 
(86°  F.-950  F.)  the  effect  of  temperature  upon  the  oxidations  of  the  body 
vanes  with  the  humidity  of  the  air.  The  greater  part  of  the  heat-loss 
at  these  high  temperatures  is  accomplished  through  the  evaporation 
of  perspiration,  and  if  the  humidity  of  the  atmosphere  be  so  great  as 
to  interfere  with  this  method  of  heat-dissipation  the  regulatory  mechan- 
isms of  the  body  become  inadequate,  the  bodily  temperature  rises  and 
with  it  the  rate  of  oxidation  and  the  total  output  of  heat,  just  as  it 


NITROGENOUS  WASTE-PRODUCTS  541 

would  in  cold-blooded  animals.  It  is  to  this  that  the  exhausting  effects 
of  the  Tropical  Climate  are  to  be  referred.  A  temperature  of  86°  F. 
in  an  atmosphere  saturated  with  moisture  is  almost  unbearable,  and 
physical  work  is,  for  Europeans  at  least,  an  impossibility,  while  a 
temperature  of  110°  F.  in  a  perfectly  dry  atmosphere  can  be  endured, 
and  even  a  considerable  amount  of  physical  work  performed,  without 
any  exceptional  discomfort,  by  persons  in  normal  health  whose  tem- 
perature-regulating mechanisms  are  in  good  order. 

Among  the  remaining  carbonaceous  waste-products  under  normal 
physiological  conditions  may  be  enumerated  Methane  which  is  derived 
from  bacterial  fermentations  in  the  intestine  but  is  exhaled  mainly 
through  the  lungs.  The  quantity  of  methane  produced  by  carnivora 
and  animals  which  subsist  upon  a  mixed  diet,  such  as  ourselves,  is 
normally  a  very  small  proportion  of  the  total  carbon  output,  but  in 
herbivora  it  may  become  very  appreciable.  Oxalic  Acid  is  regularly 
found  in  normal  urine  in  very  small  amounts,  the  normal  excretion 
being  about  0.02  grams  in  twenty-four  hours.  Its  origin  is  unknown. 
As  it  is  a  frequent  product  of  bacterial  fermentations  it  may  have  an 
alimentary  origin,  and,  again,  the  administration  of  sodium  oxalate 
leads  to  the  appearance  of  the  unchanged  oxalic  acid  in  the  urine,  and 
a  number  of  foodstuffs,  particularly  fruits  and  vegetables,  contain 
oxalates  which  would  therefore  appear  in  the  urine.  On  the  other  hand, 
the  output  of  oxalic  acid  continues  on  a  pure  protein  diet  and,  on  a 
normal  diet,  is  stated  to  be  enhanced  by  the  administration  of  con- 
siderable quantities  of  gelatin,  so  that  we  may  conjecture  that  the 
urinary  oxalic  acid  is  in  part  produced  by  the  metabolism  of  the  tissues. 
The  output  of  oxalic  acid  is  also  stated  to  be  increased  in  diabetes. 

Lactic  Acid  is  only  found  in  the  urine  in  partial  asphyxia,  or  after  the 
most  extreme  muscular  exertion;  its  appearance  in  the  urine  indicates 
imperfect  oxidation  of  carbohydrates  or  else  extraordinarily  excessive 
production  by  the  muscular  tissues. 

THE  NITROGENOUS  WASTE-PRODUCTS. 

Of  the  various  nitrogenous  waste-products,  Urea: 

/NH2 

co 


is  quantitatively  the  most  important.  The  daily  output  of  this  sub- 
stance varies  with  the  quantity  of  protein  which  is  ingested,  but  for 
the  adult  man  subsisting  upon  a  mixed  diet  the  daily  excretion  is  about 
thirty  grams,  for  a  woman  somewhat  less.  This  corresponds  to  from 
84  to  90  per  cent,  of  the  total  nitrogenous  output. 

The  quantity  of  urea  which  is  excreted  varies  directly  with  the 
quantity  of  protein  ingested.  We  have  seen  in  preceding  chapters  that 
animal  tissues  do  not  store  up  proteins  and  that  their  storage-capacity 


542  WASTE-PRODUCTS 

for  amino-acids  is  limited.  The  excess  of  amino-acids  absorbed  from 
the  intestine  is  converted  into  urea  by  a  series  of  steps  which  we  are 
about  to  discuss,  and  this  is  excreted  promptly  in  the  urine.  On  the 
other  hand  the  excretion  of  urea  upon  a  diet  low  in  proteins,  but 
abundant  in  fats  and  carbohydrates,  may  actually  be  less  than  in  star- 
vation, because  the  fats  and  carbohydrates  spare  the  tissue-protein 
from  destruction  for  the  production  of  the  energy  which  is  dissipated 
by  the  body. 

The  question  of  the  region  of  the  body  in  which  urea  originates  has 
been  the  subject  of  a  great  many  investigations.  Since  it  is  so  promi- 
nent a  constituent  of  urine,  the  kidneys  naturally  fall  first  under  sus- 
picion of  being  the  organs  in  which  the  manufacture  of  this  material 
takes  place.  This  possibility  has  been  the  subject  of  experimental 
inquiry  by  a  number  of  investigators.  If  the  kidneys  produced  urea 
to  the  extent  of  an  important  proportion  of  the  total  output,  then 
excision  of  the  kidneys  should  lead  to  the  disappearance  of  urea 
from  the  body,  or  at  any  rate  should  not  lead  to  its  accumulation. 
If,  however,  the  kidneys  simply  eliminate  urea  which  is  produced 
primarily  by  other  organs,  then  excision  of  the  kidneys  should  lead  to 
the  accumulation  of  urea  in  the  organs  and  tissue  fluids.  This- is  what 
actually  occurs,  and  the  accumulation  of  urea  under  these  circumstances 
and  in  conditions  involving  inefficient  excretion  by  the  kidneys,  as  in 
Nephritis,  has  been  repeatedly  established. 

We  must  therefore  look  elsewhere  than  to  the  kidneys  for  the  main 
source  of  the  urea  which  they  excrete.  Front  a  variety  of  different 
experimental  results  we  can  definitely  affirm  that  the  Liver  plays  a 
very  important  role  in  the  production  of  urea;  whether  it  is  the  exclus- 
ive source  of  this  substance  or  not  cannot  be  regarded  as  definitely 
established,  but  a  very  large  proportion  of  the  total  output  originates 
in  this  organ.  Thus  if  blood  be  perfused  through  the  various  organs 
in  such  a  manner  that  the  same  blood  passes  without  renewal  through 
the  tissues  over  and  over  again,  no  accumulation  of  urea  in  the  blood 
is  noted  in  the  case  of  the  kidneys  or  of  muscular  tissues,  but  a  very 
pronounced  accumulation  occurs  in  the  blood  which  is  perfused  through 
the  liver. 

The  portal  vein,  which  carries  the  blood  containing  absorbed  food- 
stuffs from  the  alimentary  wall  to  the  liver,  runs  parallel  with,  and 
very  close  to,  the  inferior  vena  cava.  By  making  an  incision  in  the 
adjoining  sides  of  these  veins  and  sewing  the  edges  together,  an  oper- 
ation which  is  known  as  Eck's  Fistula,  the  portal  circulation  is  short- 
circuited  and  the  blood  from  the  intestine,  with  its  load  of  food- 
products,  no  longer  passes  through  the  tissues  of  the  liver.  The  liver 
is,  however,  still  nourished  by  the  circulation  from  the  hepatic  artery. 
Animals  upon  which  this  operation  has  been  performed  will  survive 
for  prolonged  periods,  and  it  was  found  by  Pawlow  and  Nencki  that  in 
such  animals  the  urea  excretion  is  greatly  diminished  while  the  ammonia 
excretion  is  very  considerably  increased;  in  other  words  that  ammonia 


NITROGENOUS  WASTE-PRODUCTS  543 

to  a  certain  extent  takes  the  place  of  urea  in  the  urine  of  such  animals. 
Confirmatory  evidence  is  supplied  by  the  effects  of  degenerative 
changes  of  the  liver  upon  the  urea  output.  In  cirrhosis  of  the  liver 
and  in  the  liver-degeneration  which  is  induced  by  Phosphorus -poisoning 
there  is  a  decided  diminution  of  the  urea  output,  and  a  concurrent 
increase  in  the  ammonia  output. 

It  is  impossible  to  settle  this  question  by  extirpation  of  the  liver  in 
mammals,  since  they  do  not  survive  the  operation  for  a  sufficient 
period  to  permit  observation  of  the  excretory  products.  In  birds 
however,  this  severe  operation  may  be  performed  without  immediately 
fatal  results.  The  birds  do  not,  it  is  true,  survive  the  operation  for 
more  than  about  twenty-four  hours,  but  the  time  during  which  they 
live  is  sufficient  to  enable  us  to  ascertain  the  effect  of  the  removal  of 
the  liver  upon  the  excretory  products.  Unfortunately  urea  is  not 
the  normal  end-product  of  protein  catabolism  in  birds;  its  place  being 
taken  by  Uric  Acid,  which  forms  from  one-half  to  three-fourths  of  the 
total  nitrogenous  output.  However,  the  uric  acid  which  is  excreted  by 
birds  is  undoubtedly  the  physiological  equivalent  of  urea.  In  fact 
when  urea  is  administered  to  birds  it  is  excreted  in  the  form  of  uric 
acid,  so  that  were  the  tissues  of  birds  to  form  urea  it  would  neverthe- 
less be  excreted  in  this  form.  The  effects  of  extirpation  of  the  liver  in 
geese  were  investigated  by  Minkowski,  with  the  following  results: 

Per  cent,  of  total  nitrogen  in  the  form  of: 
Uric  acid.  Ammonia. 

Before  extirpation     ........      60  to  70  10  to  18 

After  extirpation 3  to  6  45  to  60 

These  results  are  decisive,  and  the  origin  of  at  least  ninety  per  cent, 
of  the  uric-acid  output  in  birds  must  be  in  the  tissues  of  the  liver. 
Taking  all  of  these  different  experiments  together,  therefore,  and  recol- 
lecting that  the  uric-acid  excretion  of  birds  is  the  physiological  equiva- 
lent of  the  urea  output  of  mammals,  we  are  justified  in  inferring  that 
the  liver  is  a  predominant,  if  not  the  sole  source  of  the  urea  output  of 
mammals.  Nevertheless  some  urea  output  continues  in  animals  which 
have  an  Eck  fistula,  even  when  the  hepatic  artery  is  also  ligated,  so 
that  blood  is  cut  off  altogether  from  the  liver,  and  the  output  of  urea 
is  definitely  increased  under  these  circumstances  by  the  subcutaneous 
administration  of  amino-acids.  We  can  hardly  doubt  therefore  that 
other  tissues  besides  the  liver  possess  the  power  of  manufacturing 
urea,  although  the  size  and  functional  activity  of  the  liver  enable  it 
to  play  a  predominant  role  in  this,  as  in  other  chemical  phenomena 
in  which  it  plays  a  part. 

The  question  which  next  arises  is  that  of  the  chemical  origin  or  pre- 
cursor of  urea.  A  direct  origin  from  Arginine  is  immediately  suggested 
by  mere  inspection  of  the  structural  formula  of  this  amino-acid : 

/NH2 
NH    =  C< 

\NH.CH2.CH2.CH2CH(NH2)  COOH 


544  WASTE^PRODUCTS 

and  since  the  discovery  by  Kossel  and  Dakin  of  the  existence  of  an 
enzyme,  Arginase  in  aqueous  extracts  of  the  liver,  spleen,  thymus  and 
intestinal  mucosa  which  directly  splits  arginine  with  the  production  of 
urea,  and  Ornithine : 


/NH2 

HN    =  C<  +     H20 

XNH.CH2.CH2.CH2.CH(NH2)  .COOH 
Arginine. 


-/NHj 

CO  +     CH2.(NH2)CH2.CH2.CH(NH2).COOH 


Urea.  Ornithine. 

there  can  be  no  doubt  that  a  proportion  of  the  urea  output  originates 
in  this  manner.  It  can  only  be  a  small  proportion,  however,  since 
urea  forms  over  eighty  per  cent,  of  the  total  nitrogen  output  and  only 
a  very  small  percentage  of  the  nitrogen  intake  is  in  the  form  of  arginine 
radicals. 

The  origin  of  the  greater  part  of  the  urea  output  is  undoubtedly  to 
be  traced  to  Ammonia  formed  by  deaminization  from  the  various  amino- 
acids.  We  have  seen  that  the  decrease  of  urea  output  which  accom- 
panies interference  with  the  liver-functions  also  results  in  a  correspond- 
ing increase  of  the  ammonia  output  in  the  urine,  and  this  fact  in  itself 
would  point  to  ammonia  as  a  precursor  of  urea.  It  can,  however,  be 
directly  shown  that  when  ammonia  in  the  form  of  Ammonium  Carbonate 
is  supplied  to  the  liver,  it  is  transformed  therein  into  urea.  Thus 
Nencki  and  Pawlow  have  shown  that  the  percentage  of  ammonia  con- 
tained in  the  blood  from  the  portal  vein  is  considerably  higher  than  it 
is  in  the  blood  from  the  hepatic  vein,  showing  that  the  ammonia  is 
retained  by  the  liver  as  the  portal  blood  passes  through  it.  Further- 
more, when  ammonium  carbonate  is  administered  to  animals  it  appears 
in  the  urine  as  urea,  and,  finally,  von  Schroeder  perfused  the  isolated 
liver  of  the  dog  with  ammonium  carbonate  and  obtained,  not  only  the 
retention  of  ammonia  observed  by  Nencki  and  Pawlow,  but  also  an 
actual  replacement  of  the  perfused  ammonium  carbonate  in  part  by 
urea.  Ammonium  Formate  was  similarly  transformed.  The  conversion 
of  ammonium  salts  into  urea  by  the  tissues  of  the  liver  has  therefore 
been  confirmed  in  a  variety  of  ways. 

Urea  is  the  diamide  of  carbonic  acid  and  may  be  derived  from 
carbonic  acid  by  the  successive  introduction  of  amino-groups,  an  inter- 
mediate stage  of  the  process  being  the  formation  of  Carbamic  Acid: 

/O.NH4  /NH2 


c=o  -»   c=o  —  >   c=o 

\).NH4  \NH2 

H2O  H2O 

Ammonium  carbonate.     Ammonium  carbamate.  Urea. 


NITROGENOUS  WASTE-PRODUCTS  545 

Now  it  has  been  shown  by  Macleod  and  Haskins  that  there  is  an 
equilibrium  in  aqueous  solutions  between  ammonium  carbonate  and 
Ammonium  Carbamate,  so  that  if  the  ammonium  carbamate  is  removed 
by  transformation  into  urea  a  continuous  renewal  of  the  ammonium 
carbamate  is  to  be  expected,  and  consequently  a  quantitative  conversion 
of  the  ammonium  carbonate  into  urea.  The  formation  of  ammonium 
carbamate  as  an  intermediate  product  in  the  synthesis  of  urea  in  the 
body  is  shown  by  the  fact  that  if  alkalies  be  administered  to  animals 
in  considerable  quantity  carbamates  appear  in  abundance  in  the  urine. 
A  direct  conversion  of  ammonium  carbamate  into  urea  has  been 
accomplished  by  Drechsel  by  simply  passing  an  alternating  current 
through  its  solution,  i.  e.,  by  alternate  oxidation  and  reduction  which 
is,  of  course,  equivalent  to  dehydration.  We  may  infer,  summing  up 
the  results  of  these  various  investigations,  that  ammonia,  derived  from 
amino-acids  by  the  process  of  deaminization,  is  converted  by  union 
with  carbon  dioxide  into  ammonium  carbonate,  which  spontaneously 
undergoes  partial  transformation  into  ammonium  carbamate.  The 
latter  substance  is  converted  by  alternate  oxidation  and  reduction  in 
the  liver  into  urea  which  is  subsequently  expelled  from  the  body  by  the 
kidneys.  In  Acidosis,  whether  induced  by  disordered  metabolism  or 
by  the  ingestion  of  acids  in  excess,  this  process  is  impeded  and  the 
ammonia  is  utilized  in  part  to  neutralize  the  excess  of  acids  in  the  blood 
and  tissues.  The  output  of  Ammonia  in  the  urine,  therefore,  rises  in 
acidosis  and  is,  in  fact,  a  most  valuable  means  of  detecting  and  esti- 
mating the  severity  of  that  condition. 

Next  to  urea,  but  as  a  rule  far  inferior  to  it  in  amount,  the  most 
abundant  nitrogenous  constituent  of  the  urine  is  Creatinine: 

/NH CO 

HN    =  C< 

XN(CH3).CH2 

this  substance  may  be  regarded  as  an  anhydride  of  Creatine,  or  methyl 
guanidine  acetic  acid: 

XNH2 

HN  =  c< 

XN(CH3).CH2COOH 

which,  it  will  be  remembered,  is  an  abundant  constituent  of  muscular 
tissues. 

The  daily  output  of  creatinine  in  man  is  from  1.0  to  1.7  grams  or 
from  four  to  six  per  cent,  of  the  total  nitrogenous  excretion.  Our 
views  regarding  the  probable  origin  of  creatinine  have  undergone  very 
important  modifications  in  recent  years,  thanks  to  the  fundamental 
investigations  of  Folin,  Van  Hoogenhuyze  and  Verploegh,  and  Mel- 
lanby.  It  was  formerly  assumed  without  any  doubt  that  the  source 
of  the  creatinine  in  the  urine  was  the  creatine  in  the  muscular  tissues. 
This  must  now  be  considered  to  have  become  uncertain,  and  in  any 
35 


546  WASTE-PRODUCTS 

case  we  have  come  to  attach  a  very  fundamental  significance  to  the 
creatinine  excretion  in  the  urine. 

It  was  first  pointed  out  by  Folin  that  with  varying  nitrogenous 
intakes  the  behavior  of  the  creatinine  output  is  fundamentally  different 
from  that  of  the  output  of  urea.  The  latter  rises  and  falls  almost  in 
direct  proportionality  to  the  quantity  of  protein  in  the  food.  The 
creatinine  output,  on  the  contrary,  remains  almost  unaltered  whether 
the  protein  content  of  the  diet  be  high  or  low.  The  creatinine  output 
is  not,  therefore,  derived  from  the  diet.  Thus,  for  example,  Folin 
compared  the  urea  and  creatinine  excretipn  on  a  high  protein  diet 
and  a  low  protein  diet,  with  the  following  very  striking  results: 

High  protein  diet.  Low  protein  diet. 

Volume  of  urine 1170.       c.c.  385.     c.c. 

Total  nitrogen      .      ...      .          16. 80  grams  3. 6  grams 

Urea-nitrogen       .      .      .      .      .  .   .   14.70      "  2.2 

Creatinine-nitrogen  ....  0.58      "  0.6 

The  urea  output,  it  will  be  seen,  fell  on  the  low  protein  diet  to  one- 
sixth  of  that  obtained  on  the  high  protein  diet.  The  creatinine  output, 
on  the  contrary,  remained  almost  unaltered. 

The  statement  that  the  creatinine  which  is  excreted  in  the  urine  is 
not  derived  directly  from  the  foodstuffs  must  be  qualified  to  this  extent, 
that  if  creatinine  be  contained  preformed  in  the  diet,  the  greater  part 
of  it  is  excreted  in  the  urine  unaltered  within  twenty-four  hours.  On 
the  other  hand,  if  creatine  be  administered  with  the  food  it  does  not 
appear  in  the  urine  either  in  the  form  of  creatine  or  creatinine.  In 
fact  it  usually  appears  to  be  excreted  by  some  other  channel  or  else 
retained  by  the  body,  for  Folin  in  many  instances  administered  crea- 
tine without  causing  any  increase  even  in  the  total  nitrogen  of  the 
urine.  It  has  been  suggested  by  Mellanby  that  bacteria  in  the  intestine 
decompose  the  creatine  and  retain  it  in  their  tissues.  However  this 
may  be,  these  observations  render  it  certain  that  the  creatine  which  is 
contained  preformed  in  a  meat-diet  is  not  the  source  of  the  creatinine 
in  the  urine. 

Since  the  output  of  creatinine  is  so  extraordinarily  independent  of 
fluctuations  in  the  diet,  Folin  regards  it  as  originating  in  the  Endog- 
enous Metabolism  of  the  tissues  themselves,  while  a  great  part  of  the 
urea  arises  from  the  destruction  by  deaminization  of  amino-acids 
which  have  never  become  part  of  the  living  protoplasm  of  the  body, 
and  therefore  represents  a  product  of  Exogenous  Metabolism.  The 
exogenous  metabolism  rises  and  falls  with  the  intake  of  foodstuffs,  but 
the  endogenous  metabolism  persists  practically  unchanged  under  a 
variety  of  nutritional  conditions.  It  represents  the  "wear  and  tear" 
or  irreversible  spontaneous  decomposition  of  the  tissues. 

It  is  questionable,  however,  whether  the  creatinine  output  represents 
the  endogenous  metabolism  of  the  whole  body  or  whether  it  does  not, 
on  the  contrary,  arise  from  the  endogenous  metabolism  of  the  muscular 


NITROGENOUS  WASTE-PRODUCTS  547 

tissues  only.  The  daily  output  of  creatinine,  although  so  constant 
in  a  given  individual,  varies  in  different  individuals  with  the  weight, 
and  more  especially  with  the  degree  of  muscular  development.  Obese 
persons,  notwithstanding  their  high  body-weight,  have  a  low  creatinine 
output,  wliile  comparatively  lean  persons,  who  by  virtue  of  muscular 
development  have  a  like  weight,  exhibit  a  high  creatinine  output. 
It  is  true  that  muscular  work  on  a  normal  diet  does  not  increase  the 
creatinine  output,  but  then  we  have  seen  that  on  a  normal  mixed  diet 
the  muscles  do  not  derive  their  energy  from  the  metabolism  of  their 
own  substance  (protein)  but  from  the  oxidation  of  carbohydrates. 
When,  however,  muscular  work  is  performed  during  starvation,  the 
creatinine  output  is  definitely  increased.  In  other  words  the  actual 
destruction  of  muscular  tissue  results  in  an  increase  of  creatinine 
excretion. 

It  appears  very  probable  that  the  normal  products  of  the  disinte- 
gration of  tissue-protein  are  similar  to  or  identical  with  the  substrates 
out  of  which  tissue-protein  is  synthesized,  namely,  the  amino-acids, 
for  we  have  seen  that  the  process  of  tissue-synthesis  is  a  balanced 
reaction  which  is  retarded  by  its  products,  and  this  can  only  be  true 
if  the  products  of  the  synthesis  break  down,  in  the  first  place,  into 
the  substances  which  form  the  substrates  of  the  forward  reaction.  The 
amino-acids  which  are  thus  set  free  are  cast  into  the  general  stock  of 
circulatory  and  storage  amino-acids,  undergo  their  share  of  exogenous 
metabolism  or  deaminization,  and  participate  with  the  ingested  amino- 
acids  arising  from  the  foodstuffs  in  determining  the  Nutrient-level  of 
the  tissue-fluids.  If  the  nutrient-level  falls,  as  in  starvation,  the  amino- 
acids  of  tissue  origin  form  a  large  proportion  of  the  whole  mass  of  cir- 
culating amino-acids,  and  their  deaminization  results  in  a  continual 
drainage  which,  in  turn,  results  in  a  steady  loss  of  tissue-substance. 
There  must,  in  fact,  be  an  endogenous  or  tissue-source  of  urea,  for 
otherwise  urea  excretion  would  ultimately  fall  to  zero  in  starvation, 
which  it  never  does.  In  fact,  even  in  starvation  the  urea  output  still 
exceeds  very  decidedly  the  creatinine  output.  On  the  other  hand,  if 
the  tissues  must  use  their  own  substance  for  the  performance  of 
external  work,  at  any  rate  in  muscular  tissues,  the  breakdown  of  the 
protein  or  of  amino-acids  resulting  therefrom  takes  another  course, 
with  the  production  of  creatinine.  The  effect  of  this  must  be  to  initiate 
a  process  analogous  to  repair  or  Regeneration  by  the  resynthesis  of 
the  lost  tissue-proteins  from  amino-acids. 

Creatine  is  not  a  normal  constituent  of  the  urine  of  adult  men  and, 
as  has  been  stated  above,  the  ingestion  of  creatine  leads  to  no  increase 
in  the  creatinine  output,  nor  does  it  lead  to  the  appearance  of  any. 
creatine  in  the  urine.  In  the  urine  of  women,  on  the  contrary,  creatine 
is  found  during  menstruation  and  after  delivery,  and  the  ingestion 
of  creatine  leads  to  the  appearance  of  a  small  proportion  of  the  creatine 
in  the  urine.  In  the  urine  of  children  creatine  is  a  regular  constituent. 
According  to  Krause  it  disappears  from  the  urine  of  boys  at  about  five 


548  WASTE-PRODUCTS 

or  six  years  of  age,  but  persists  in  the  urine  of  girls  until  puberty.  The 
ingestion  of  creatine  in  children  is  also  followed  by  an  increase  in  the 
creatine  output  in  the  urine.  The  adult  has  therefore  acquired  a  power 
of  destroying  or  utilizing  creatine  which  is  imperfect  in  women  and  only 
slightly  developed  in  young  children. 

Apart  from  the  question  of  the  nature  of  the  tissues  in  which  creatine 
and  creatinine  originate  we  have  to  consider  the  problem  of  the  chemi- 
cal precursors  or  parent-substances  from  which  they  originate.  A  very 
obvious  possibility  is  that  they  may  arise  from  Arginine. 

/NH2 
HN   =  C 

\NH.CH2.CH2.CH2.CHNH2.COOH 

by  breaking  the  hydrocarbon-chain  and  methylation  of  one  of  the  nitro- 
gens in  the  guanidine  nucleus.  It  has  been  stated  that  creatine  may 
arise  from  proteins  in  the  autolytic  decomposition  of  tissues  in  the 
absence  of  bacteria  but  no  other  evidence  of  its  formation  from  arginine 
has  yet  been  adduced. 

Creatinine  is  a  reducing  agent  and  decolorizes  cupric  hydroxide  in 
alkaline  solutions,  but  does  not  precipitate  cuprous  oxide  as  the  reduc- 
ing sugars  do.  It  is  precipitated  by  Picric  Acid,  but  if  treated  with 
picric  acid  in  alkaline  solutions  it  yields  a  red  coloration  which  turns 
yellow  upon  the  addition  of  acids  (Jaffe's  Reaction).  If  an  alkaline 
solution  is  treated  with  Sodium  Nitroprusside  the  mixture  turns  ruby 
red  (Weyl's  Reaction)  and  then  yellow.  If  this  yellow  solution  is  treated 
with  excess  of  acetic  acid  and  boiled,  it  becomes  first  green  and  then 
blue  (Salkowskfs  Reaction).  JafiVs  reaction  is  utilized  by  Folin  for 
the  colorimetric  estimation  of  creatinine  in  urine.  Creatine  is  esti- 
mated by  converting  it  into  creatinine  by  boiling  with  dilute  acid  and 
then  reestimating  the  creatinine. 

Uric  Acid  is  an  exceedingly  important  constituent  of  the  urine,  since 
it  represents,  in  man,  the  end-product  of  the  purine  metabolism.  The 
average  output  per  day  on  a  mixed  diet  is  0.7  grams,  and  the  ratio  of 
uric  acid  to  urea  varies  between  1  : 50  and  1  : 70. 

Uric  acid  is  derived  from  the  Purine  Bases  by  oxidation;  it  is  2,  6,  8, 
trioxypurine : 

HN CO 

I         I 

i     i 

OC         C— NH 


HN € NH/ 

It  may  be  prepared  synthetically  from  urea  and  glycocoll.  On 
heating  in  sealed  tubes  with  hydrochloric  acid,  glycocoll,  carbon 
dioxide  and  ammonia  are  produced.  It  is  capable  of  acting  as  a  weak 
acid  and  forms  two  series  of  salts,  the  Monourates,  containing  one,  and 


NITROGENOUS  WASTE-PRODUCTS  549 

the  Diurates,  containing  two  molecules  of  base.  The  so-called  quadri- 
urates  are  non-existent. 

Uric  acid  yields  a  variety  of  characteristic  color  reactions,  among 
which  the  Murexide  Test,  already  described  in  connection  with  the 
purine  bases,  must  be  included.  Uric  acid  is  a  reducing  agent  and 
reduces  an  alkaline  cupric  hydroxide  solution;  the  quantity  of  uric 
acid  which  is  present  in  urine  is,  however,  insufficient  to  produce  an 
appreciable  precipitation  of  cuprous  oxide.  If  a  drop  of  uric  acid  dis- 
solved in  sodium  carbonate  be  placed  upon  a  filter-paper  moistened 
with  silver  nitrate  solution,  reduction  occurs  with  the  production  of  a 
yellow  or  brown  spot  (Schiff's  Reaction).  If  a  weak  alkaline  solution  of 
uric  acid  in  water  is  treated  with  a  soluble  zinc  salt  a  white  precipitate 
is  produced  which  gradually  turns  blue  if  exposed  to  light  and  air,  or 
immediately,  if  treated  with  sodium  persulphate  (Ganassini's  Reaction). 
With  a  certain  mixture  of  phosphoric  and  phosphotungstic  acids  uric 
acid  yields  a  blue  coloration  (Folin  and  Macallum's  Reaction),  the 
origin  of  which  is  unknown. 

The  elimination  of  uric  acid  is  definitely  increased  by  a  diet  which 
contains  excess  of  purines  or  of  Nucleic  Acids.  This  is  due  to  the  fact 
that  the  adenine  and  guanine,  split  off  from  the  nucleic  acids,  are 
transformed  in  the  tissues  into  Hypcxanthine  and  Xanthine,  by  the 
deaminizing  enzymes  adenase  and  guanase.  The  hypoxanthine  is  sub- 
sequently converted  into  xanthine  and  the  xanthine  into  uric  acid 
by  a  specific  oxidizing  enxyme  which  is  found  in  a  variety  of  animal 
tissues,  and  is  designated  Xanthine-oxidase : 

HN— CO  HN— CO  HN— CO 

!     I  I     I  II 

HC     C— NH\  OC    C— NH\  OC     C— NH, 

J|      ||  \CH  +     O    ->         |       ||  VJH  +     O    -*         |       ||  >CO 

N_C_  N  +  HN— C—  N  *  HN— C— NH/ 

Hypoxanthine.  Xanthine.  Uric  acid. 

Nevertheless,  the  elimination  of  uric  acid  continues  on  a  purine  or 
nuclein-free  diet.  In  a  series  of  experiments  on  himself  and  others, 
Folin  was  able  to  reduce  the  daily  elimination  to  0.3  grams  on  a  diet 
of  cream  and  starch,  but  this  minimum  could  not  be  reduced.  Evi- 
dently, therefore,  there  is,  as  in  the  case  of  amino-acids  and  other 
foodstuffs,  an  Endogenous  Metabolism  of  purines  as  contrasted  with  an 
Exogenous  Metabolism.  That  the  endogenous  metabolism  represents 
the  actual  breaking  down  of  tissues  is  shown  by  the  fact  that  if  destruc- 
tion of  tissue  is  remarkably  augmented,  as  in  pneumonia,  leukemia, 
or  in  severe  burns,  the  uric  acid  excretion  rises  decisively. 

There  is  no  evidence  that  mammalian  tissues  can  synthesize  uric 
acid  from  any  other  source  than  purines.  It  is  true  that  the  elimination 
of  uric  acid,  and  of  purine  bases  also,  is  increased  by  an  increase  in  the 
dietary  intake,  but  this  is  true  whether  the  increase  be  nitrogenous  or 
non-nitrogenous,  and  it  follows  very  rapidly  upon  the  intake  of  food. 


Time 
10  to  11                             ...... 

Urea,  grains. 
.     ."1.07 

11  to  12       
12  to     1 

.      .      1.13 
.      1  .  07 

1  to    2  (meai  at  1.30)     
2to    3       

.      0.64 
.      1.12 

3to    4       
4to    5       .      .      .      .   '  
5  to    6                                    .... 

.      .      1.16 
.      .      0.84 
.      .      1.16 

6  to    7                              .            ... 

.      .      1.20 

7  to  .  8       '. 
8  to    9       
9  to  10      
10  to  11 

.      .      1.37 
.      .      1.47 
.      1.33 
1.33 

550  WASTE-PRODUCTS 

Thus  Hopkins  and  Hope,  after  fasting  for  six  hours,  consumed  a  meal 
of  bread  and  potatoes,  pratically  purine-free,  with  the  following  results: 

Uric  acid, 
milligrams. 

26 
27 
24 
21 
22 
38 
40 
56 
39 
30 
33 
24 
23 

Thus  a  slight  rise  in  the  urea  output  occurred  about  six  hours  after 
the  ingestion  of  the  food,  and  continued  for  some  time,  but  a  sharp 
rise  in  the  uric  acid  output  occurred  within  two  hours,  and  the  excre- 
tion fell  tc  nearly  the  normal  value  again  before  the  urea  excretion 
began  to  rise.  It  is  not  known  where  this  uric  acid  originates,  but 
it  would  appear  to  be  manifestly  connected  with  the  activities  of  the 
alimentary  canal,. and  to  be  endogenous  in  origin.  It  is  for  this  reason 
that  the  uric  acid  and  purine  output  is  greater  during  the  day  than 
it  is  at  night. 

In  birds  and  .reptiles  the  relationships  are  quite  different.  These 
possess  the  power  of  synthesizing  uric  acid,  most  probably  from 
Ammonia  and  Lactic  Acid,  since,  if  the  liver  be  extirpated  in  birds,  the 
place  of  the  uric  acid  in  the  excreta  is  taken  by  ammonia,  and  large 
amounts  of  lactic  acid  are  excreted  concurrently.  An  increase  of  uric 
acid  elimination  in  birds  follows  the  administration  of  lactic  acid  and 
other  hydroxy-acids  and  dibasic  acids  of  the  aliphatic  series.  This 
power  is,  however,  lacking  in  the  mammalia. 

In  the  majority  of  mammals,  uric  acid  is  not  the  end-product  of 
the  purine  metabolism,  but  undergoes  in  part  or  almost  wholly,  trans- 
formation into  Allantoin  which  is  excreted  in  the  urine: 

HN co  H2N 

oc 

+     H20     +     O      = 

HN 

.This  transformation,  which  is  known  as  Uricolysis,  is  brought  about  by 
an  oxidizing  enzyme,  Uncase,  which  occurs  in  tissue-extracts  prepared 
from  the  liver,  kidney  and  other  organs.  It  trantforms  uric  acid  almost 
quantitatively  into  allantoin.  It  is  probable,  however,  that  the  destruc- 
tion of  uric  acid  does  not  stop  at  this  stage  but  proceeds  further  and, 
ultimately,  to  the  formation  of  urea  and  other  products.  Thus  Ascoli 


NITROGENOUS  WASTE-PRODUCTS 


551 


and  Izar  have  shown  that  if  an  extract  of  liver  which  has  completely 
destroyed  a  given  sample  of  uric  acid  in  the  presence  of  oxygen  be 
excluded  from  oxygen,  the  uric  acid  is  gradually  reformed.  This  is 
what  one  would  expect  if  we  had  here  to  deal  with  a  reversible  oxidation. 
The  curious  feature  of  their  results  is,  however,  that  the  addition  of 
allantoin  had  no  effect  upon  the  production  of  uric  acid,  appearing  to 
indicate  that  the  production  of  allantoin  was  not  an  intermediate  step 
in  the  resynthesis. 

The  power  of  uricolysis  is  absent  from  the  tissues  of  man  and  the 
chimpanzee  —  a  fact  which  would  have  gladdened  the  heart  of  Huxley, 
could  he  but  have  known  it.  All  other  mammals,  so  far  as  we  know, 
contain  uricase  in  their  tissues.  The  following  results,  cited  after 
Hunter  and  Givens,  show  the  relative  proportions  of  uric  acid  and 
allantoin  in  the  urine  of  various  mammals.  The  "Uritolytic  Index" 
is  the  proportion,  expressed  as  a  percentage  of  uric  acid,  which  has 
been  converted  by  the  animal  into  allantoin. 


Orders  and  species. 
Marsupialia: 

Opossum   . 
Rodentia: 

Rabbit 

Guinea-pig 

Rat      .      . 
Ungulata: 

Sheep  . 

Goat    . 

Cow     . 

Horse  . 

Pig.      . 
Carnivora : 

Raccoon 

Badger 

Dog      . 

Coyote 
Primates : 

Monkey    . 

Chimpanzee 

Man     , 


Total  purine 

nitrogen, 

gms. 

.      0.04 


0.2  to  0.6 
1.0 
8.0 
1.6 
0.3 


.      0.25 
O.ltoO.3 
.      0.15 

.     0.045 
... 
.     0.2 


Percentage  of  purine-allantoin- 
nitrogen. 


Allantoin.       Uric  acid. 
76.0  19.0 


91.0 
93.7 

64.0 
81.0 
92.1 
88.0 
92.3 

92.6 
96.9 
97.1 
95.6 

66.0 
2.0 


6.0 
3.7 

16.0 
7.0 
7.3 

12.0 

1.8 

5.4 
1.9 
1.9 
2.6 

8.0 
90.0 


Bases. 
6.0 


3.0 

2.7 

20.0 

12.0 

0.7 

0.5 

5.8 

2.0 
1.2 
1.3 

1.8 

26.0 
8.0 


Uricolytic 
index. 


79 

95 
94 
96 

80 
92 
93 

88 
98 

95 
98 
98 
97 

89 
0 
2 


Allantoin  has  been  isolated  by  Hunter  from  the  blood  of  the  ox,  pig, 
horse  and  sheep,  but  could  not  be  detected  in  the  blood  of  man. 

It  is  not  by  any  means  certain,  however,  notwithstanding  the 
inability  to  convert  uric  acid  into  allantoin,  that  the  tissues  of  man 
cannot  destroy  uric  acid  in  some  other  manner.  Thus,  Taylor  and 
Rose  fed  a  human  subject  for  three  days  on  a  diet  very  low  in  purines, 
namely  milk,  eggs,  starch,  and  sugar.  For  three  days  following,  a 
part  of  the  protein,  namely  three  grams  per  day  out  of  a  total  of  ten 
was  given  in  the  form  of  "sweetbread"  nitrogen  (sheep's  pancreas). 
For  four  days  succeeding  this  twice  as  much  "sweetbread"  nitrogen 
was  given,  namely  six  out  of  ten  grams,  and  this  was  succeeded  by  a 


552  WASTE-PRODUCTS 

period  of  four  additional  days  on  a  purine-free  diet.    The  following 
were  the  results  obtained: 

1st  period,  4th  period, 

purine-free  diet.        2d  period.  3d  period.  purine-free  diet. 

Total  urinary  N  .      .      .  8.9  8.7  9.1  8.80 

UreaN  +  NH3  .      .      .  7.3  7.1  7.1  7.05 

CreatinineN        .      .      .  0.58  0.55  0.56  0.47 

Purine  N  (total)         .      .  0.11  0.17  0.26  0.10 

Uric  acid 0.09  0.14  0.24  0.07 

Undetermined  N       .      .  0.91  0.88  1.18  1.18 

The  intake  of  purine  nitrogen  in  the  second  period  was  0.17  and 
in  the  third  0.34  grams  per  day,  so  that  the  increased  output  only 
accounted  for  one-half  of  the  intake.  The  purine  was  not  simply 
stored,  to  be  excreted  later,  for  as  soon  as  the  purine-rich  diet  ceased 
the  excretion  fell  to  the  figure  previously  obtained  on  a  purine-free  diet. 
The  only  alternatives  that  remain  are  either  that  part  of  the  purine 
was  never  absorbed  from  the  intestine  or  else  that  the  tissues  of  the 
subject  destroyed  the  purines  in  some  manner  which  did  not  result  in 
the  formation  of  uric  acid  or  allantoin.  We  may  recall  the  observa- 
tions of  Ascoli  and  Izar,  cited  above,  which  tend  also  to  the  conclusion 
that  there  are  means  of  destroying  uric  acid  in  the  tissues  which  do 
not  involve  the  production  of  allantoin  as  an  intermediate  stage. 

In  persons  afflicted  with  Gout  deposits  of  uric  acid  form  in  various 
tissues  and  particularly  in  the  joints.  The  origin  of  these  deposits  has 
been  the  subject  of  much  investigation.  There  is  a  definite  increase  in 
the  uric-acid  content  of  the  blood  in  such  persons,  although  the  uric- 
acid  output  in  the  urine  is  not  above  the  normal.  Evidently,  there- 
fore, the  kidneys  are  functioning  abnormally  and  in  such  a  way  as  to 
constitute  a  barrier  to  the  excretion  of  uric  acid.  The  limiting  con- 
centration in  the  blood  at  which  transmittal  through  the  renal  epi- 
thelium begins  is  raised,  and  hence  the  uric  acid,  dammed  back  in  the 
blood,  accumulates  therein.  This  alone,  however,  is  not  a  sufficient 
cause  of  gout,  for  uricemia  occurs  also  in  nephritis,  and  in  lead  poison- 
ing, without  the  production  of  gouty  deposits.  It  has  been  suggested 
that  the  solubility  of  uric  acid  in  the  blood  is  diminished  in  gouty 
persons,  but  no  positive  evidence  of  this  has  been  advanced.  The 
origin  of  the  tendency  of  uric-acid  deposits  to  form  in  the  joints  when 
they  do  occur  at  all  is,  however,  rendered  clear  by  the  fact  upon  which 
emphasis  is  laid  by  Taylor,  that  Cartilage,  possibly  owing  to  its  high 
content  of  sodium  salts,  diminishes  the  solubility  of  sodium  urate  in 
water,  so  that  deposits  are  precipitated  upon  it  from  saturated  solu- 
tions. 

The  solubilities  of  the  monourates  of  potassium,  sodium  and  ammo- 
nium at  37°  C.  in  water  have  been  determined  by  Gudzent  as  follows: 

Salt  of  uric  acid.  Solubility  in  grams  per  liter. 

Potassium t     t     2.7002 

Sodium 1.5043 

Ammonium 0.7413 


NITROGENOUS  WASTE-PRODUCTS  553 

The  solubility  of  sodium  urate  in  blood  is,  however,  no  less  than  three 
times  its  solubility  in  water  (Taylor).  This  is  not  due  to  the  formation 
of  diurates,  since  at  the  reaction  of  the  blood  diurates  cannot  exist. 
The  nature  of  the  factor  which  so  greatly  increases  the  solubility  of 
uric  acid  is  unknown. 

It  was  formerly  considered  possible  to  remove  uric  acid  from  the 
body  by  administering  Alkalies,  the  assumption  being  that  the  greater 
alkalinity  of  the  blood  resulted  in  the  formation  of  the  more  soluble 
diurates.  We  now  know  that  the  alkalinity  of  the  blood  is  only 
increased  to  an  almost  imperceptible  extent  by  this  means  and  that  the 
maximum  alkalinity  attainable  would  not  suffice  to  form  diurates,  or 
indeed  to  influence  perceptibly  the  solubility  of  uric  acid.  Nevertheless, 
the  administration  of  certain  alkalies  may  be  assumed  to  facilitate  the 
solution  of  uric  acid  by  the  formation  of  a  certain  proportion  of  the 
more  soluble  potassium  salt,  or  of  the  Lithium  Urate  which  is  the  most 
soluble  salt  of  uric  acid. 

The  most  remarkable  effect  upon  the  elimination  of  uric  acid  is, 
however,  that  of  phenylquinoline-carbonic  acid  or  Atophan: 

CH        c— COOH 

\ 

CH 
C— C«H6 

v 

CH         N 

The  administration  of  this  substance  and  of  other  quinoline-carbonic 
acid  derivatives  has  been  shown  by  Nicolaier  to  increase  the  amount 
of  uric  acid  excreted  by  the  kidneys  to  an  extraordinary  extent,  even 
to  twice  or  three  times  the  normal  amount.  No  other  physiological 
effects  are  noted  and  no  other  constituent  of  the  urine  is  altered  in 
amount.  The  increased  elimination  occurs  on  a  purine-free  diet  and 
has  been  shown  by  Folin  and  Lyman  to  be  accompanied  by  a  fall  in 
the  uric  acid  content  of  the  blood.  In  other  words  the  hyperexcretion 
of  uric  acid  is  due  to  the  increased  permeability  of  the  kidneys  for  this 
substance,  just  as  the  glycosuria  following  phloridzin  administration 
is  due  to  increased  permeability  of  the  kidneys  for  glucose.  The 
hyperexcretion  does  not  persist  if  the  administration  be  continued, 
the  daily  output  sinking  within  a  few  days  to  only  slightly  above  the 
normal  level,  probably  because  the  available  supply  of  urates  in  the 
blood  and  tissue-fluids  has  become  exhausted.  There  is,  however,  a 
continuous  slight  hyperexcretion  throughout  a  prolonged  period  of 
administration,  and  when  nuclear  tissues  are  administered  in  the  diet 
a  greater  proportion  of  uric  acid  is  excreted  in  consequence  than  is 
usually  the  case.  The  formation  of  uric  acid  from  the  nucleic  acids  is 
thus  facilitated  by  atophan,  but  this  effect  is  probably  only  a  secondary 


554  WASTE-PRODUCTS 

one,  depending  upon  the  reduction  of  the  concentration  of  the  urates 
in  the  tissue-fluids,  and  the  tendency  of  the  tissue-enzymes  to  spon- 
taneously reestablish  the  normal  equilibrium  between  the  blood  and 
the  tissues. 

The  only  amino-acid  which  normally  occurs  in  urine  is  Glycocoll, 
or  amino-acetic  acid,  which,  in  very  small  amounts,  appears  to  be  a 
constant  constituent.  If,  however,  an  excess  of  leucine  or  alanine  be 
introduced  into  the  circulation  they  will  appear  in  the  urine.  It  would 
appear  that,  normally,  deaminization  and  utilization  are  too  rapid  to 
permit  of  the  accumulation  of  amino- acids  in  the  blood  in  sufficient 
amount  to  cause  elimination  by  the  kidneys.  If,  however,  the  rate  of 
deaminization  be  slowed,  as,  for  instance,  in  degenerative  changes  of 
the  liver  induced  by  chloroform-necrosis  or  phosphorus-poisoning,  then 
a  variety  of  amino-acids  may  appear  in  the  urine.  It  is  also  stated  by 
Loewy  that  the  amino-acid  content  of  the  urine  is  increased  at  high 
altitudes. 

When  the  urea,  creatinine,  uric  acid  and  glycocoll  of  the  urine  are 
added  together,  there  is  always  a  considerable  remainder  of  nitrog- 
enous excretion.  Part  of  this  arises  from  the  sulphur-containing  and 
conjugated  excreta  which  are  about  to  be  described,  part  is  stated  by 
Abderhalden  and  Pregl  to  be  present  in  the  form  of  Polypeptides  which 
yield  glycocoll,  leucine,  alanine,  glutamic  acid  and  phenylalanine  on 
hydrolysis.  When  all  the  nitrogen  in  hitherto  defined  substances  is 
summed  up,  however,  there  is  still  a  small  remainder  which,  although 
it  arises  from  substances  excreted  in  small  amount,  may  nevertheless 
be  of  physiological  importance.  It  is  derived  in  part  from  exogenous 
and  in  part  from  endogenous  metabolism. 

CONJUGATED  EXCRETA. 

A  variety  of  substances  occur  in  the  urine  which  arise  from  the 
union  of  a  genuine  excretory  product  with  another  molecule  which 
serves  as  a  vehicle  to  accomplish  its  elimination.  Such  excreta  are, 
for  example,  the  Conjugated  Glucuronic  Acids  which  are  normally  pres- 
ent in  the  urine  in  small  amounts  and  are  greatly  augmented  by  the 
ingestion  of  certain  poisons,  of  which  a  partial  list  has  been  given  in  a 
previous  chapter  (Chapter  III).  The  function  of  the  glucuronic  acid 
moiety  of  the  molecule  appears  to  be  in  the  main  to  render  harmless 
the  associated  substance  which  is  usually  of  a  toxic  character.  Only 
definite  classes  of  toxic  substances  are  eliminated  in  this  manner, 
however. 

The  Glucuronates  which  normally  occur  in  the  urine  are  in  the  main 
the  phenyl,  indoxyl  and  skatoxyl  glucuronates,  the  latter  two  in  very 
small  amounts,  The  phenol,  indoxyl  and  skatoxyl  radicals  are  derived, 
it  is  believed,  mainly  from  putrefactive  decomposition  of  aromatic 
amino-acids,  particularly  tyrosine  and  tryptophane,  by  the  intestinal 
bacteria.  These  substances  are  in  themselves  very  toxic,  but  their 


CONJUGATED  EXCRETA  555 

conjugates  with  glucuronic  acid  are  harmless.  Upon  boiling  with 
dilute  acids  or  occasionally  even  on  allowing  urine  to  stand,  they 
decompose,  setting  free  glucuronic  acid  and  the  associated  radical  of 
the  conjugate. 

The  origin  of  the  glucuronic  acid  in  urine  is  unknown.  The  most 
natural  assumption  is  to  suppose  that  the  toxic  substances  which  are 
eliminated  in  this  way  combine  in  the  body  with  glucose,  and  that  the 
oxidation  of  glucose  is  by  this  so  hindered,  that  it  only  proceeds  as 
far  as  the  conversion  of  the  primary  alcohol-group  into  a  carboxyl- 
group.  Certainly  the  phenyl-glucuronic  acid  is  a  compound  of  the 
glucoside  type,  i.  e.,  the  phenyl  radical  is  attached  to  the  glucu- 
ronic acid  by  the  aldehyde-group.  On  the  other  hand  if  camphor  be 
administered  in  large  amounts  to  phloridzinized  dogs,  although  the 
excretion  of  glucuronates  is  very  greatly  increased  thereby,  the  excre- 
tion of  glucose  is  either  not  diminished  at  all  or  only  slightly  diminished, 
a  fact  which  would  appear  to  indicate  some  other  source  than  glucose 
for  the  glucuronic  acid. 

A  very  important  excretory  conjugate  is  the  conjugated  sulphuric 
acid,  indoxyl-sulphuric  acid  or  Indican: 

C.O.SOzOH 

/\  -•'•-• 

C6H4  CH 


NH 

which  yields  Indigo  when  treated  with  oxidizing  agents.  This  substance 
arises  by  conjugation  of  indoxyl  with  sulphuric  acid  and  is  the  form  in 
which  the  greater  part  of  the  indoxyl  output  is  present  in  the  urine. 
The  indoxyl  output  varies  with  the  extent  of  putrefactive  processes 
in  the  intestine.  Any  measure  of  Intestinal  Stasis,  such  as  that  induced 
by  tying  off  a  loop  of  small  intestine,  results  in  an  increase  of  the 
indican  output.  The  subcutaneous  injection  of  indol  leads  to  an 
increased  output  of  indican,  while  the  administration  of  an  excess  of 
Tryptophane  in  this  way  does  not.  Evidently  the  tissues  do  not  decom- 
pose tryptophane  in  such  a  way  as  to  liberate  indole,  while  the  intes- 
tinal bacteria,  like  the  majority  of  putrefactive  bacteria,  generate  a 
large  proportion  of  indole  from  tryptophane,  which,  after  absorption 
is  oxidized  to  indoxyl  and  then  excreted  in  the  form  indicated  above. 
It  must  be  remembered  that  the  indican  output,  although  generally 
running  parallel  with  the  degree  of  intestinal  stasis  or  putrefaction,  is 
not  a  reliable  measure  of  intestinal  putrefaction  when  taken  by  itself, 
for  the  output  depends,  not  solely  upon  putrefaction,  but  also  upon 
the  proportion  of  tryptophane  which  is  contained  in  the  proteins  of 
the  diet.  Thus,  if  a  large  part  of  the  protein  intake  be  supplied  by 
Gelatin,  which  contains  no  tryptophane,  the  indican  output  becomes 
very  small  although  putrefactive  processes  may  not  be  diminished  in 


556  WASTE-PRODUCTS 

the  slightest  degree.  Then,  again,  even  upon  a  standard  diet,  the  out- 
put of  indican  may  be  expected  to  vary  greatly  with  the  type  of  infect- 
ing organisms  in  the  intestine.  Thus  Herter  has  shown  that  Bacillus 
coli  communis  produces  indole  but  only  traces  of  skatole,  which  is 
the  methyl  derivative  of  indole,  while  certain  anaerobic  putrefactive 
bacteria  produce  skatole,  in  preference  to  indole,  from  tryptophane. 
Skatol  does  not  appear  to  be  normally  excreted  in  the  urine,  at  least 
in  the  form  of  a  conjugated  sulphuric  acid. 

Phenol-sulphuric  Acid  and  Cresol-sulphuric  Acid  are  constant  con- 
stituents of  urine,  and,  as  in  the  case  of  indican,  the  output  is  obviously 
derived  from  the  products  of  intestinal  putrefaction.  It  is  probable 
that  these  substances,  of  which  the  total  excretion  may  amount  to 
fifty  milligrams  per  day,  originate  from  the  putrefactive  decomposi- 
tion of  Tyrosine  and  Phenylalanine. 

In  general  it  may  be  said  that  while  aliphatic  alcohols,  terpenes  and 
many  phenols  are  excreted  in  the  urine  in  conjugation  with  glucuronic 
acid,  the  greater  part  of  the  phenols  and  polyphenols  are  excreted  in 
conjugation  with  sulphuric  acid.  Yet  a  third  vehicle  of  excretion  is 
that  afforded  by  conjugation  with  Glycocoll,  or  ammo-acetic  acid. 
Thus  Benzole  Acid,  appears  in  the  urine  after  administration  in  the 
form  9f  the  conjugated  Hippuric  Acid  : 


•   C6H6COOH     +     CftNHzCOOH      =     C6H5COHNCH2COOH     +     H2O 
Benzole  acid.  Glycocoll.  Hippuric  acid. 

Hippuric  acid  is  a  very  abundant  constituent  of  the  urine  in 
Herbivora,  comparatively  scanty  in  the  urine  of  Carnivora,  and  inter- 
mediate in  amount  in  the  urine  of  partially  herbivorous  animals  like 
ourselves.  The  daily  excretion  in  man,  subsisting  upon  a  normal 
mixed  diet,  is  about  0.7  grams,  but  after  eating  quantities  of  vegeta- 
bles or  fruits  it  may  rise  as  high  as  2  grams. 

The  synthesis  of  hippuric  acid  from  benzoic  acid  and  glycocoll  is 
accomplished  within  the  tissues  of  the  kidneys  themselves.  This,  in 
fact,  was  the  first  synthetic  process  which  was  definitely  shown  to  take 
place  in  animal  tissues  (by  Schmiedeberg  and  Bunge)  and  also  the 
first  to  be  performed  by  admixture  of  the  components  of  the  reaction 
with  macerated  tissue.  It  is  not  improbable,  however,  that  some 
measure  of  hippuric  acid  synthesis  may  also  occur  in  other  organs. 

When  large  amounts  of  benzoic  acid  are  administered  to  animals  the 
elimination  of  glycocoll  is  far  in  excess  of  the  glycocoll  which  could  be 
obtained  by  simple  hydrolysis  of  the  protein.  "  Thus  McCollum  and 
Hoagland  brought  a  pig  into  a  condition  of  minimal  nitrogen  metab- 
olism by  administering  a  diet  of  starch  containing  75  calories  per 
kilogram  body-weight.  To  this  diet  was  then  added  varying  amounts 
of  benzoic  acid,  and  finally  hydrochloric  acid  and  benzoic  acid  were 
given  together.  The  total  nitrogenous  output  and  its  partition  among 
the  various  nitrogenous  fractions  in  the  urine  were  determined  ifr  the 
different  periods  of  the  experiment  with  the  following  results: 


CONJUGATED  EXCRETA 


557 


Period. 

No.  of 

days. 

Food. 

Total 

N. 

Urea 

N. 

NH3 

N. 

Creatinine 

N. 

Hippuric 
acid  + 
other  N. 

I 

12 

Starch,  75  cal.  per  kilo  + 

2.56 

1.43 

0.21 

0.488 

0.424 

alkali  salts 

II 

4 

Same  +  4  g.  benzoic  acid 

2.63 

1.29 

0.21 

0.456 

0.681 

III 

7 

Same  +  1  0  g.  benzoic  acid 

2.23 

0.58 

0.22 

0.484 

0.948 

IV 

5 

Same  +  16g.  benzoic  acid 

2.86 

0.55 

0.38 

0.437 

1.492 

V 

5 

Same  +  16  g.  benzoic  acid 

4.03 

0.54 

1.44 

0.424 

1.632 

+  10  g.  of  25  per  cent. 

HC1 

It  will  be  seen  that  despite  the  great  increase  of  hippuric  acid  excre- 
tion induced  by  these  large  dosages  of  benzoic  acid  the  total  daily 
nitrogen  elimination  was  unaffected.  Evidently  body-protein  was  not 
attacked  to  provide  the  glycocoll  needful  for  the  synthesis  of  the  hip- 
puric acid.  The  glycocoll  was  evidently  derived  at  the  expense  of  the 
urea-fraction,  and  the  endogenous  catabolism,  in  so  far  as  it  is  repre- 
sented by  the  creatinine  output,  remains  unaffected.  On  the  other 
hand  the  acidosis  induced  by  hydrochloric  acid  resulted  in  a  large 
increase  of  the  total  nitrogen  output,  the  chief  part  of  the  increase 
being  Ammonia  which  performs  the  protective  function  of  neutralizing 
a  part  of  the  excess  of  acid.  The  urea  and  creatinine  output  were  alike 
unaffected  by  the  administration  of  the  acid. 

The  glycocoll  moiety  of  hippuric  acid  must  therefore  be  traced  to 
the  same  origin  as  urea,  and  this,  it  will  be  remembered,  is  the  amino- 
acids  of  the  tissue-fluids.  No  less  than  thirty-five  per  cent,  of  the 
nitrogen  of  the  food  may  be  excreted  as  hippuric  acid,  and  no  protein 
contains  this  percentage  of  glycocoll.  It  is  evident  that  glycocoll  may 
be  synthesized  from  other  amino-acids.  It  might  be  imagined  that 
the  benzoic  acid  unites  with  other  amino-acids  which  thereafter  under- 
go partial  oxidation  until  only  the  residue  of  glycocoll  is  left.  Injec- 
tion of  such  compounds  synthetically  prepared,  however,  leads  to  no 
increase  in  the  hippuric  acid  output.  It  seems  probable,  therefore, 
that  glycocoll  may  form  a  normal  disintegration-product  of  many 
amino-acids,  that  under  ordinary  circumstances  it  is  finally  deaminized, 
but  that  when  toxic  substances  that  will  pair  with  it,  namely  aromatic 
acids,  are  present  in  the  tissue-fluids,  deaminization  is  prevented  by 
the  conjugation. 

The  power  of  the  tissues  to  synthesize  glycocoll  is  of  very  great 
importance,  since  it  not  only  enables  the  body  to  protect  itself  against 
such  poisons  as  benzoic  acid,  but  also  enables  suckling  animals  to 
synthesize  their  tissue-proteins  from  a  protein  which  is  totally  lacking 
in  glycocoll,  namely  the  casein  of  milk. 

When  the  administration  of  benzoic  acid  is  pushed  beyond  the 
limit  of  the  glycocoll  available  from  the  proteins  of  the  diet  the  protec- 
tive mechanism  breaks  down  and  free  benzoic  acid  appears  in  the 
urine.  Under  no  circumstances,  it  appears,  are  tissue-proteins  attacked 


558  WASTE-PRODUCTS 

for  this  purpose  nor  are  the  proteins  of  the  blood  broken  down  to  fur- 
nish glycocoll,  for  the  ratio  of  albumins  (containing  no  glycocoll)  to 
globulins  in  the  blood-serum  remains  unaltered  by  benzoic-acid 
administration. 

In  the  metabolism  of  birds,  Ornithine,  or  diami  no  valeric  acid  plays 

CH2(NH2)CH2.CH2.CH(NH2)COOH 

the  part  which  is  taken  by  glycocoll  in  the  metabolism  of  mammals, 
or,  at  all  events,  to  the  extent  of  being  the  substance  utilized  to 
detoxicate  and  eliminate  benzoic  acid.  The  conjugated  acid  which 
appears  in  the  urine  of  birds  when  benzoic  acid  is  administered  to 
them  is  Ornithuric  Acid,  which  splits  into  benzoic  acid  and  ornithine 
when  it  is  hydrolyzed. 

AROMATIC  OXYACIDS. 

The  putrefaction  of  proteins  in  the  intestine  results  in  the  formation 
of  Paraoxyphenylacetic  Acid  and  Paraoxyphenylpropionic  Acid  as  inter- 
mediate stages  in  the  decomposition  of  tyrosine,  and  they  pass  in 
small  amounts  unchanged  into  the  urine. 

It  has  been  observed,  from  the  middle  ages,  that  human  urine  in 
certain  very  rare  instances  may  regularly  darken  on  exposure  to  air 
and  ultimately  turn  black.  The  individuals  exhibiting  this  peculiarity, 
which  is  designated  Alcaptonuria,  are  very  rare,  and  yet  the  condition 
constitutes  a  definite  peculiarity  of  metabolism  which  has  often  been 
described,  and  has  been  very  carefully  investigated.  The  darkening 
is  due  to  the  spontaneous  oxidation  of  dioxyphenyl  acetic  acid  or 
Homogentisic  Acid: 

CH 

/\      .    ;  .    :i:;i 

HC  COH 

HOC  CH 


CH 

CH2 
I 
COOH 

which  is  a  constituent  of  the  urine  of  these  persons.  The  individuals 
who  display  this  peculiarity  do  not  appear  to  suffer  any  inconvenience 
from  it,  and  cases  only  reach  the  physician  through  the  alarm  created 
by  the  extraordinary  appearance  of  the  urine  after  standing,  or  by 
failure  to  secure  an  insurance-policy,  for  dioxyphenyl  acetic  acid  is  a 
reducing-substance  and  may  be  reported  by  a  physician  who  is  unfa- 
miliar with  the  typical  indications  of  the  disease,  as  glycosuria.  The 


WASTE-PRODUCTS  OF  THE  SULPHUR  METABOLISM      559 

reduction  of  cupric  hydroxide  solution  by  the  urine  of  an  alcaptonuric 
individual  is,  however,  accompanied  by  darkening  or  even  blackening 
of  the  fluid,  so  that  no  confusion  of  diagnosis  should  be  possible  even 
on  superficial  observation. 

The  homogentisic  acid  in  alcaptonuria  arises  from  the  tyrosine  and 
phenylalanine  radicals  in  the  proteins  of  the  food.  If  the  diet  contains 
little  tyrosine  or  phenylalanine  the  output  sinks,  if  much  it  rises. 
The  administration  of  tyrosine  or  phenylalanine  by  mouth,  or  of 
glycyltyrosine  hypodermically,  leads  to  quantitative  excretion  of  the 
aromatic  nucleus  in  the  form  of  homogentisic  acid.  The  elimination 
continues,  although  it  is  reduced,  in  starvation,  and  this,  together 
with  the  fact  that  it  may  be  enhanced  by  subcutaneous  or  intravenous 
administration  of  the  parent  acids,  shows  that  the  homogentisic  acid 
is  not  derived  from  intestinal  cleavage  or  putrefaction. 

Evidently  the  alcaptonuric  is  unable  to  complete  the  oxidation  of 
the  aromatic  nuclei  of  Tyrosine  and  Phenylalanine,  just  as  the  Diabetic 
is  unable  to  complete  the  oxidation  of  /3-oxybutyric  acid.  Curiously 
enough,  however,  as  Garrod  and  Neubauer  have  shown,  tryptophane 
is  normally  utilized  by  persons  who  display  alcaptonuria. 

According  to  Garrod  there  is  but  one  degree  of  alcaptonuria  and  that 
is  complete.  Either  the  excretion  of  homogentisic  acid  amounts  to 
several  grams  a  day  or  it  is  absent  from  the  urine,  and  usually  the 
condition  is  present  from  earliest  childhood.  It  is  evidently  the  exog- 
enous metabolism  only  of  tyrosine  and  phenylalanine  which  is  affected 
for  no  defect  of  development  or  loss  of  weight  in  the  adult  occurs 
such  as  we  would  expect  to  happen,  were  tissue-protein  destroyed  to 
produce  the  homogentisic  acid.  It  is  the  circulating  amino-acids,  which 
normally  undergo  complete  combustion  after  deaminization,  which  are 
the  source  of  this  substance. 

It  is  probable  that  homogentisic  acid  represents  a  normal  inter- 
mediate product  in  the  oxidation  of  the  oxyphenyl-oxypropionic  acid 
which  results  from  the  deaminization  of  tyrosine.  The  curious  fea- 
ture of  the  transformation,  however,  resides  in  the  fact  that  whereas 
tyrosine  has  only  one  hydroxyl-group  in  the  benzene  nucleus  and  that 
in  the  para  position,  homogentisic  acid  has  two,  one  in  the  ortho  and 
the  other  in  the  meta  position.  It  is  found,  however,  that  this  is  the 
only  class  of  dioxyphenols  which  is  oxidized  by  normal  persons,  other 
dioxyphenols  being  excreted  in  the  form  of  conjugates  in  the  urine. 
The  alcaptonuric  therefore  differs  from  the  normal  person  in  that  his 
inability  to  oxidize  diphenols  extends  to  the  single  class  which  normal 
individuals  can  oxidize,  namely  those  in  which  the  hydroxyl-groups 
occupy  the  ortho  and  meta  positions  relatively  to  the  side-group. 

WASTE-PRODUCTS  OF  THE  SULPHUR  METABOLISM. 

The  waste-products  of  the  sulphur  metabolism  are  of  three  types, 
namely  Inorganic  Sulphates,  Ethereal  or  Conjugated  Sulphates  and  the 


560  WASTE-PRODUCTS 

Neutral  Sulphur  compounds  in  which  the  sulphur  is  not  present  as  a 
sulphuric  acid  radical. 

These  three  fractions  have  been  found  by  Folin  to  vary  in  a  char- 
acteristic manner  with  the  abundance  of  proteins  in  the  diet.  On  high 
and  low  protein  diets  respectively  the  following  daily  output  of  the 
various  sulphur-containing  excreta  was  observed: 

Protein-rich  diet.  Protein-poor  diet. 

Volume  of  urine       .  1170  c.c.  385  c.c. 

Total  nitrogen    .      .  16.80gm.  3.60gm. 

Total  sulphur  (SO«)  3.64     "  0.76     " 

Inorganic  SO3     .      .  3.27     "    (90. 0  per  cent.)  0.46     "    (60.5  per  cent.) 

Ethereal  SO3       .      .  0.19     "    (  5 . 2  per  cent.)              0.10     "    (13.2  per  cent.) 

Neutral  SO3        .      .  0.18     "    (  4 . 8  per  cent.)             0.20    "    (26.3  per  cent.) 

It  will  be  observed  that  a  reduction  of  the  total  sulphur  output 
to  one-fifth,  reduced  the  output  of  inorganic  sulphates  to  one-seventh, 
and  of  ethereal  sulphates  to  one-half,  while  the  output  of  neutral 
sulphur  remains  unaltered.  Folin  draws  an  analogy  between  the 
neutral  sulphur  output  and  the  creatinine  output  among  the  nitroge- 
nous excreta,  and  regards  the  neutral  sulphur  as  originating  from  the 
degeneration  of  tissue-protein,  the  Endogenous  Metabolism,  while  the 
inorganic  sulphates  represent  the  extent  of  Exogenous  Metabolism  or 
the  destruction  of  circulating  amino-acids  which  have  not  become 
constituents  of  living  tissue. 

The  ethereal  sulphates,  representing  conjugated  phenols,  indican 
and  so  forth,  have  usually  been  regarded  as  indicative  of  the  extent  of 
Intestinal  Putrefaction.  The  relatively  slight  degree  to  which  they  are 
reduced  by  a  reduction  of  protein  intake  to  one-half  is  adduced  by 
Folin  as  an  indication  that  they  may  possibly  arise  from  the  endog- 
enous metabolism  of  tissues.  It  is  to  be  noted,  however,  as  Hopkins 
has  pointed  out,  that  we  have  no  right  to  assume  that  Intestinal  Putre- 
factions are  reduced  proportionately  to  the  reduction  of  the  protein 
intake.  On  the  contrary,  the  proportion  of  the  protein  intake  which 
reaches  the  lower  intestine  without  absorption  is  as  a  rule  very  small, 
unless  it  chances  to  be  a  form  of  protein  which  is  indigestible,  such  as 
raw  egg-albumen,  or  which  contains  a  large  glycocoll-fraction,  such  as 
gelatin.  A  large  proportion  of  the  putrefaction  in  the  lower  intestine 
must  be  attributed  to  the  protein  contained  in  the  mucous  secretions 
of  the  intestine  itself.  Thus  Whipple  has  shown  that  toxic  proteoses 
of  bacterial  origin  may  be  absorbed  from  an  isolated  loop  of  intestine, 
from  which  the  contents  have  previously  been  removed.  Hence 
reduction  of  the  protein  intake  only  reduces  one,  and  not  necessarily 
the  larger  source  of  intestinal  putrefaction,  and  the  reduction  of 
ethereal  sulphates  to  one-half,  by  a  reduction  of  protein  intake  to  one- 
fifth,  is  probably  the  utmost  that  could  be  expected.  We  may,  there- 
fore, ascribe  to  the  ethereal  sulphates,  as  to  the  inorganic  sulphates,  a 
primarily  exogenous  origin. 

Another  channel  of  sulphur  excretion  is  the  Bile,  wherein  sulphur 


WASTE-PRODUCTS  OF  THE  SULPHUR  METABOLISM      561 

is  contained  in  the  form  of  Taurine,  which,  combined  with  cholic  acid, 
forms  the  taurocholic  acid  fraction  of  the  mixed  bile-acids.  Taurine 
is  amino-ethyl  sulphuric  acid,  and  its  relationship  to  the  sulphur- 
containing  amino-acid  of  the  tissue-proteins,  Cystine,  is  shown  in  the 
following  formulae : 

CH2— S— S— CH2  CH2(SO2OH) 

CHNH2          CHNH2  CH2NH2 

I  I 

COOH  COOH 

Cystine.  Taurine. 

The  taurine  thus  excreted  is  mainly  reabsorbed  and  either  reexcreted 
as  taurocholic  acid  or  else  transformed  into  products  which  are  elim- 
inated in  the  urine.  It  will  be  observed  that  the  relationship  of  taurine 
to  cystine  is  a  very  simple  one,  decarboxylation  and  oxidation  of  the 
sulphur  serving  to  convert  the  cystine  into  taurine.  This  being  the 
case  it  is  of  very  great  interest  to  note  that  the  excretory  products  to 
which  these  compounds  give  rise  are  very  diverse,  for  as  Salkowski 
originally  showed,  and  his  results  have  been  confirmed  and  amplified 
by  Schmidt,  von  Adelung  and  Watson,  the  administration  of  taurine 
in  large  doses  to  man  by  mouth,  or  subcutaneous  or  intravenous 
injection,  leads  to  a  large  increase  in  the  Neutral  Sulphur  output,  over 
eighty  per  cent,  of  the  taurine  being  excreted  within  twenty-four 
hours  in  a  "neutral"  form  which  Salkowski  has  identified  as  Tauro- 
carbamic  Acid.  Now  the  administration  of  cystine  in  moderate  dosage, 
or  of  polypeptides  containing  cystine,  leads  to  an  increase  in  the  inor- 
ganic sulphates  only,  and  a  very  large  dosage  is  required  to  elicit  an 
increase  of  neutral  sulphur. 

The  fact  that  the  administration  of  cystine,  whether  by  mouth  or 
intravenously,  results  in  an  increased  output  of  inorganic  sulphates 
suggests  that  a  portion  of  the  endogenous  sulphur  metabolism  may 
be  represented  in  the  inorganic  sulphates,  for,  as  we  have  previously 
argued  in  connection  with  a  possible  endogenous  origin  of  urea,  if  the 
circulating  amino-acids  stand  in  equilibrium  with  the  tissue-amino- 
acids,  as  the  results  of  Van  Slyke  indicate,  and  these  latter  in  equi- 
librium with  the  tissue-proteins,  then  the  disintegration-products  of 
tissue-proteins  must  be  the  amino-acids  themselves,  for  otherwise 
protein  synthesis  would  go  on  indefinitely  and  unchecked.  But  the 
amino-acids,  including  cystine  of  course,  when  once  released  from 
the  tissues  must  be  thrown  into  the  common  supply  and  undergo  their 
share  of  exogenous  metabolism.  Indeed  it  may  be  questioned  whether 
the  neutral  sulphur  output  really  represents  the  metabolism  of  cystine 
in  the  tissues  of  the  body  considered  collectively,  or  whether  it  does  not 
possibly  represent  the  destruction  of  a  special  fraction  of  the  cystine 
which  is  converted  by  the  liver  into  taurine,  and  a  series  of  products 
obtained  from  the  sulphur-containing  compounds  of  the  nervous 
system,  cartilage,  etc.,  in  which  sulphur  is  present  in  radicals  other 
36 


562  WASTE-PRODUCTS 

than  cystine.  Among  the  constituents  of  the  neutral  sulphur  fraction 
may  be  enumerated  Sulphocyanides  which  are  found  in  traces  in  the 
urine  and  also  in  the  Saliva,  Chondroitin-sulphuric  Acid,  and  a  number 
of  poorly-defined  nitrogenous  acids  which  have  been  designated  the 
Oxyproteic  Acids. 

In  rare  instances  Cystine  is  found  to  occur  in  the  urine  in  notable 
quantities,  as  much  as  0.5  to  1.5  grams  being  excreted  in  one  day. 
This  condition,  known  as  Cystinuria,  is  a  much  more  serious  abnor- 
mality than  alcaptonuria,  which  it  resembles  in  being  due  to  a  defect 
of  metabolism,  because  the  large  excretion  of  this  sparingly  soluble 
amino-acid  often  leads  to  the  formation  of  deposits  or  calculi  in  the 
bladder.  According  to  Garrod,  cystinuria  is  a  rarer  disease  than 
alcaptonuria,  but  it  reaches  the  physician  more  frequently  because 
of  the  serious  natiire  of  the  symptoms  which  arise.  The  failure  to 
oxidize  cystine,  which  is  characteristic  of  the  cystinuric  patient,  fre- 
quently extends  to  other  amino-acids,  and  amines,  such  as  Cadaverine 
and  Putrescine,  derived  from  the  decarboxylation  of  Lysine  and  Orni- 
thine  may  also  appear  in  the  urine,  and  occasionally,  leucine  and 
tyrosine.  In  such  cases  cystinuria  is  evidently  an  expression  of  a 
general  defect  of  the  deaminizing-mechanism. 

An  experimental  cystinuria  may  be  induced  in  animals  by  the 
administration  of  halogen-benzenes,  such  as  monochlorbenzene  or 
monobrombenzene.  The  halogen-benzene  is  paired  with  cystine  and 
excreted  in  this  form  as  Mercapturic  Acid,  in  combination  with  glu- 
curonic  acid.  The  excretion  of  cystine  in  these  cases  is  accompanied 
by  a  diminution  of  the  output  of  inorganic  sulphates. 

The  presence  of  cystine  in  the  urine  may  be  suspected  if  hexagonal 
crystals  are  deposited  which  are  soluble  in  ammonia  and  insoluble  in 
acetic  acid.  If  a  few  crystals  are  dried,  placed  on  a  slide  and  covered 
with  a  cover-glass  underneath  which  is  introduced  a  drop  of  strong 
hydrochloric  acid,  as  each  crystal  is  touched  by  the  acid  a  cluster  of 
fine  prisms  is  seen  to  spring  from  it,  consisting  of  cystine  hydrochloride 
(Wollaston's  Test). 

In  passing  it  may  be  stated  that  the  Phosphorus  of  the  diet  is  wholly 
or  almost  wholly  excreted  in  the  form  of  phosphates  in  the  urine  and 
the  feces. 

URINARY  PIGMENTS. 

A  variety  of  urinary  pigments  have  been  described  by  different 
investigators,  but  only  three  pigments  have  been  definitely  character- 
ised. These  are  Urochrome,  a  pigment  to  which  the  yellow  color  of 
urine  is  mainly  due,  Urobilin  which  is  voided  in  the  form  of  a  colorless 
chromogen,  Urobilinogen,  which  is  converted  into  urobilin  by  exposure 
to  air  under  the  influence  of  light,  and  Uroerythrin,  which  is  frequently 
but  not  invariably  present. 

On  saturating  urine  with  ammonium  sulphate,  urochrome  remains 
in  solution  while  urobilin  is  precipitated.  When  a  solution  of  urobilin 


PROPERTIES  AND  COMPOSITION  OF   URINE  563 

is  dissolved  in  ammonia  and  a  little  zinc  chloride  solution  is  added 
the  mixture  turns  red  with  a  green  fluorescence;  urochrome,  on  the 
contrary,  does  not  yield  fluorescent  solutions. 

Both  of  these  pigments  are  closely  related  to  the  bile-pigments  and, 
therefore,  to  hemoglobin.  They  yield  the  pyrrole  reactions  and 
strongly  resemble  substances  which  are  obtainable  from  Bilirubin  by 
reduction.  Urobilin,  or  its  parent-substance  urobilinogen  is  a  con- 
stant constituent  of  the  feces,  but  before  the  identity  of  the  two  pig- 
ments was  realized  the  urobilin  in  the  feces  received  a  separate  name, 
Stercobilin.  The  quantity  of  these  pigments  in  the  urine  is  distinctly 
increased  in  all  fevers,  also  in  hemorrhage  and  in  conditions  involving 
the  destruction  of  red  blood-corpuscles,  and  in  diseases  of  the  liver. 

Uroerythrin  is  the  pigment  which  frequently  gives  a  red  color  to 
urinary  sediments,  particularly  to  sediments  of  uric  acid,  which,  owing 
to  its  presence,  may  appear  like  grains  of  cayenne  pepper.  It  does  not 
yield  fluorescent  solutions  and  is  rapidly  decolorized  by  light.  The 
normal  color  of  solutions  is  pink,  but  strong  sulphuric  acid  changes 
this  to  carmine,  and  alkalies  to  green.  Uroerythrin  is  believed  not  to 
be  related  to  bilirubin  but  to  be  derived  from  Skatole.  The  quantity  is 
increased  by  muscular  activity,  profuse  perspiration,  alcohol,  immod- 
erate eating,  fevers  and  diseases  of  the  liver. 

The  presence  of  urobilinogen  in  the  feces  and  the  probable  deriva- 
tion of  uroerythrin  from  skatole  render  an  alimentary  origin  of  these 
pigments  very  probable.  It  is  likely  that  urochome  and  urobilin  arise 
by  bacterial  decomposition  of  the  bile-pigments  in  the  lower  intestine. 
In  confirmation  of  this  view  it  is  found  that  strong  Intestinal  Putre- 
faction leads  to  an  increase  of  the  urobilin  output  while  exclusion  of 
bile  from  the  intestine  reduces  the  output  to  zero.  If  the  exclusion  of 
bile  from  the  intestine  be  due  to  mechanical  occlusion  of  the  bile-ducts, 
then  bile-pigments,  but  not  urobilin,  appear  in  the  circulation  and  in 
the  urine. 

THE  PROPERTIES  AND  COMPOSITION  OF  URINE. 

The  volume  of  the  urine  which  is  voided  daily  necessarily  varies 
very  greatly  with  the  quantity  of  water  which  is  drunk,  the  quantity 
of  water  contained  in  the  food,  the  amount  of  fluid  lost  from  the  body 
by  perspiration  and  a  variety  of  other  factors  such  as  the  presence  or 
absence  of  Diuretics  such  as  Caffein  or  Theobromin  in  the  diet,  or  hyper- 
activity  of  the  posterior  lobe  of  the  pituitary  body  which  may  lead  to 
a  chronic  hyper  secret  ion  of  a  dilute  urine  containing  no  sugar;  a  con- 
dition known  as  Diabetes  Insipidus. 

The  Specific  Gravity  of  the  urine  necessarily  varies  with  its  volume, 
usually  fluctuating  between  1.008  and  1.030.  The  reaction  is  usually 
acid,  but  immediately  after  a  meal  an  alkaline  reaction,  the  "alkaline 
tide"  may  frequently  be  observed,  and  on  a  purely  vegetable  diet 
the  urine  is  not  infrequently  alkaline.  The  sulphur  and  phosphorus 


564  WASTE-PRODUCTS 

in  the  proteins  of  a  meat-diet  are  oxidized  wholly  or  in  part  to  the 
highly  dissociated  sulphuric  and  phosphoric  acids  which  decrease  the 
alkali-reserve  of  the  blood  and  tissues  and  are  excreted  as  acid  salts  in 
the  urine,  while  the  alkaline  salts  in  vegetables  are  oxidized  to  carbon- 
ates or  bicarbonates  and  excreted  as  such. 

According  to  Fitz  and  Van  Slyke  the  titratable  acidity  of  the  urine 
(employing  phenolphthalein  as  an  indicator)  runs  remarkably  parallel, 
in  conditions  of  Acidosis,  with  the  decrease  of  the  alkali-reserve.  In 
order  to  observe  this  parallelism,  however,  we  must  add  to  the  titrat- 
able acidity  the  amount  of  Ammonia  in  the  urine  which  has  been 
furnished  by  the  tissues  as  a  means  of  neutralizing  a  portion  of  the 
excess  of  acid.  This  can  be  estimated  by  the  method  of  Sorensen,  the 
Formol  Titration,  which  depends  upon  the  fact  that  formaldehyde  in 
faintly  alkaline  solutions  unites  with  ammonia  to  form  hexamthylene- 
tetramine,  which  has  a  neutral  reaction : 

4NH4C1  +  6  HCHO  +  4  NaOH      =     N4(CH2)6  +   10  H2O  +  4  NaCl 

The  urine  is  first  rendered  very  faintly  alkaline  to  phenolphthalein, 
then  neutral  formaldehyde  is  added  and  the  quantity  of  alkali  which 
must  be  added  to  render  the  urine  alkaline  again  is  determined  by 
titration.  This  is  equivalent  to  the  ammonia  which  has  been  converted 
into  hexamethylene-tetramine.1 

The  relationship  observed  by  Fitz  and  Van  Slyke  is  expressed  by 
them  in  the  following  formula,  which  is  an  adaptation  of  the  formula 
of  Ambard  for  the  excretion  of  urea  and  chlorides: 


Bicarbonates  in  the  plasma      =     80  — 


where  D.is  the  titratable  acidity  plus  the  ammonia  output,  W  the 
weight  of  the  individual  and  C  the  concentration  of  acids  in  the  urine, 

or   •-,  where  V  is  the  volume  of  urine.     The  figure  80  represents  the 

maximum  yield  of  carbon  dioxide  in  volumes  per  cent,  which  may  be 
obtained  by  treating  blood-serum  with  sulphuric  acid.  Reduction  of 
the  alkali-reserve  below  this  point  results  in  the  urinary  excretion  of 
an  excess  of  acid  radicals  which  is  expressed  by  the  factor: 


This  relationship  is  purely  empirical  and  the  agreement  between  the 
calculated  and  observed  values  of'  the  alkali-reserve  cannot  be  relied 

1  The  — NH»  groups  of  amino-acids  will  react  with  formaldehyde  in  the  same  way 
as  ammonia.  The  concentration  of  amino-acids  in  the  urine  is  so  small,  however,  that, 
as  a  rule  it  may  be  neglected. 


NORMAL  COMPOSITION  OF   URINE  565 

upon  to  within  ten  per  cent.  It  nevertheless  is  of  value  as  serving  to 
show  that  titratable  acidity  of  the  urine,  if  added  to  the  ammonia,  or 
protective  basic  output,  is  a  real  indication  of  the  presence  or  absence 
of  acidosis. 

We  have  seen  that  the  diurnal  output  of  most  of  the  nitrogenous 
excreta  is  profoundly  influenced  by  the  diet.  No  normal  composition 
of  the  urine  can  therefore  be  formulated  which  is  not  subject  to  wide 
fluctuations  which  are  nevertheless  within  the  limits  of  diversity  which 
may  be  exhibited  by  a  single  normal  individual  under  varying  dietary 
conditions.  The  following  may,  however,  serve  to  illustrate  the  com- 
position to  which  the  urine  of  a  normal  individual  subsisting  upon  a 
moderate  and  mixed  diet  would  more  or  less  closely  approximate : 

% 

NORMAL  COMPOSITION  OF  URINE. 

(Illustrative  Analysis.) 

The  following  represents  a  normal  twenty-four-hour  sample  of 
urine  of  volume  1500  c.c.  and  specific  gravity  1.010-1.015: 

Constituent.  Weight  in  Approximate 

grams.  percentage. 

Water .  1440.0  96.0 

Solids 60.0  4.0 

Urea 35.0  2.33 

Uric  acid 0.75  0.05 

Hippuricacid 0.7  C.05 

Oxalic  acid 0.015  0.001 

Aromatic  oxy-acids 0.06  0.004 

Creatinine 1.0  0.07 

Thiocyanic  acid  (as  KSCN)       ....  0.15  0.01 

Indican 0.01  0.001 

Ammonia 0.65  0.04 

Sodium  chloride 16.5  1.10 

Phosphoric  acid  (P2O6) 2.5  0.15 

Total  sulphuric  acid 2.5  0.15 

Silicic  acid 0.45  0.03 

Potassium  (K2O) 2.5  0.15 

Sodium  (Na2O) 5.0  0.30 

Calcium  (CaO) 0.25  0.015 

Magnesium  (MgO) 0.30  0.02 

Iron 0.005  0.0004 

REFERENCES. 

THE  CARBONACEOUS  WASTE-PRODUCTS: 

Pembrey:     Jour.  Physiol.,  1901,  27,  p.  66;  1903,  29,  p.  195. 

Johansson:     Skand.  Arch.%.  Physiol.,  1901,  11,  p.  273. 

Hdri:     Pfliiger's  Arch.,  1909,  130,  p.  112. 

Warburg:     Ergeb   d.  Physiol.,  1914,  14,  p.  253. 

Krogh:     The  Respiratory  Exchange  in  Animals  and  Man,  London,  1916. 

MacLeod:     Physiology  and  Biochemistry  in  Modern  Medicine,  St.  Louis,  1918. 

Lusk:     The  Science  of  Nutrition,  Philadelphia,  1919. 
THE  NITROGEINOUS  WASTE-PRODUCTS: 

Abel  and  Muirhead:     Arch.  f.  exp.  Path.  u.  Pharm.,  1893,  32,  p.  467. 

Hopkins  and  Hope:     Jour.  Physiol.,  1898-99,  23,  p.  271. 

Kossel  and  Dakin:     Zeit.  f.  physiol.  Chem.,  1904,  41,  p.  321;  1904,  42,  p.  181. 

Macleod  and  Haskins:     Jour.  Biol.  Chem.,  1905-6,  1,  p.  319. 


566  WASTE-PRODUCTS 

THE  NITROGENOUS  WASTE-PRODUCTS: 

Van  Hoogenhuyze  and  Verploegh:     Zeit.  physiol.  Chem.,  1905,  46,  p.  415;  1908,  57, 
p.   161;   1909,  59,  p.   101. 

Folin:     Am.  Jour.  Physiol.,  1905,  13,  p.  66;  Jour.  Am.   Med.  Assn.,   1914,  63,  p. 
823. 

Mellanby:     Jour.  Physiol.,  1907-8,  36,  p.  447. 

Ascoli  and  Izar:     Zeit.  f.  physiol.  Chem.,  1908-9   58,  p.  529;  1909,  62,  p.  347. 

Mendel:     Ergeb.    d.  Physio!  ,  1911,  11,  p.  418. 

Taylor:     Digestion  and  Metabolism,  Philadelphia,  1912. 

Dakin:     Oxidations  and  Reductions  in  the  Animal  Body,  London,  1912. 

Taylor  and  Rose:     Jour.  Biol.  Chem.,  1913,  14,  p.  419. 

Hunter  and  Givens:     Ibid.,  1914,  18,  p.  403. 

Hunter,  Ibid.,  1916-17,  28,  p    369. 

Denis  and  Minot:     Ibid.,  1917,  31,  p.  561. 
CONJUGATED  EXCRETA: 

Hopkins.     Guy's  Hospital  Gazette,  1907,  21,  p.  424. 

McCollum  and  Hoagland.     Jour.  Biol.  Chem.,  1913-14,  16,  p.  321. 

Jolles:     Zeit.  physiol   Chem.,  1915,  94,  p.  79. 

Sherwin:     Jour.  Biol.  Chem.,  1917,  31,  p.  307. 

Dubin:     Ibid.,  1917,  31,  p.  255. 

OXYACIDS  AND  SULPHUR  DERIVATIVES: 

Folin:     Vide  supra. 

Garrod:     Inborn  Errors  of  Metabolism,  London,  1909. 

Schmidt,  von  Adelung  and  Watson:     Jour.  Biol.  Chem.,  1918,  33,  p.  501. 
PIGMENTS: 

Garrod:     Jour.  Physiol.,  1894-95,  17,  pp.  349  and  439;  1897,  21,  p.  190. 

Garrod  and  Hopkins:     Ibid.,  1896,  20,  p.  112. 
ACIDITY  OF  URINE: 

Fitz  and  Van  Slyke:     Jour.  Biol.  Chem.,  1917,  32,  p.  495. 


PART  VI. 

THE  ENERGY-BALANCE  OF  THE  ORGANISM, 


CHAPTER  XXIII. 
THE  ANIMAL  BODY  AS  A  MACHINE. 

THE  APPLICABILITY  OF  THE  LAW  OF  THE  CONSERVATION  OF 
ENERGY  TO  LIVING  ORGANISMS. 

To  all  of  our  not  very  remote  forebears  and  to  the  majority  of  those 
of  our  contemporaries  who  vote,  legislate  and  govern  in  this  our  present 
day,  Life  was,  or  is,  a  thing  apart  from  the  Universe,  independent  of 
cosmic  laws,  controlling  rather  than  expressing  the  forces  of  nature. 
The  inversion  of  this  primitive  idea  which  was  ultimately  to  result  in 
the  attainment  of  our  present  conception  of  life,  as  the  outcome  of 
forces  which  it  does  not  of  itself  create,  originated  in  the  investigations 
of  that  greatest  of  French  chemists,  Lavoisier. 

The  clue  to  the  true  nature  of  the  processes  of  combustion  had 
previously  been  provided  by  the  discovery  by  Priestley  that  air  con- 
tains a  substance  which  is  essential  to  combustion  and  is  consumed 
thereby.  It  was  Lavoisier,  however,  who  showed  that  this  gas  is 
absorbed  by  and  becomes  combined  with  the  burning  substance,  and 
the  amplification  of  this  discovery  led  to  the  enunciation  of  the  law 
of  the  Conservation  of  Matter.  The  corresponding  law  in  the  domain 
of  energy-transformation  was  not  formulated  until  1845,  over  fifty 
years  later.  Nevertheless  it  is  to  Lavoisier  also  that  we  must  accredit 
the  investigations  which  first  established  the  applicability  of  the  law 
of  the  Conservation  of  Energy  to  animals.  It  has  frequently  happened 
in  the  history  of  scientific  investigation,  that  a  truth  which  was  not 
generally  apprehended  or  clearly  enunciated  at  the  time  has  never- 
theless been  tacitly  assumed  in  advance  of  their  period  by  investi- 
gators possessing  exceptional  powers  of  insight  and  discovery.  It  is  a 
mistake  to  suppose  that  successful  scientific  discovery  is  the  outcome  of 
purely  logical  processes  of  thought  in  the  mind  of  the  investigator. 
The  great  discoverer  appears  to  be  distinguished  from  equally  diligent 
but  less  successful  investigators  quite  as  much  in  his  possession  of  a 


568  THE  ANIMAL  BODY  AS  A   MACHINE 

species  of  intuitive  sympathy  with  the  order  of  nature,  as  in  his  purely 
intellectual  endowments  as  these  are  ordinarily  understood.  There 
can  be  no  question  at  all  that  both  Lavoisier  and  Faraday,  without 
ever  having  formulated  it  in  so  many  words,  and  certainly  without 
adequate  proof  of  its  validity,  nevertheless  assumed  the  truth  of  the 
law  of  the  conservation  of  energy  and  were  guided  in  their  investiga- 
tions by  this  assumption. 

Lavoisier  had  shown  in  1790  that  the  oxygen  absorbed  and  trans- 
formed into  other  substances  by  a  man  or  animal  is  increased  by  the 
performance  of  Muscular  Work  and  by  exposure  to  a  low  temperature. 
Work  and  the  production  of  Bodily  Heat  were  thus  correlated  with  the 
occurrence  of  chemical  reactions  which  were  known  to  liberate  energy, 
i.  e.,  combustions.  The  next  step  was  to  institute  a  direct  comparison 
between  the  heat  of  combustion  of  a  carbonaceous  material  and  the 
heat-evolution  of  an  animal,  a  comparison  which  has  since  then  been 
repeated  many  times,  and  with  ever-increasing  exactitude.  The 
material  chosen  by  Lavoisier  as  a  standard  for  comparison  was  pure 
carbon.  He  measured  the  amount  of  heat  evolved  in  the  conversion 
of  the  carbon  into  carbon  dioxide,  and  he  then  measured  the  amount 
of  heat  and  carbon  dioxide  given  off  by  a  guinea-pig  in  a  period  of 
ten  hours.  The  heat-evolution  was  estimated  from  the  latent  heat  of 
ice  which  was  melted  by  the  heat  of  the  burning  carbon  in  the  one 
experiment  and  by  the  heat  of  the  animal's  body  in  the  other.  It  was 
found  that  the  guinea-pig  communicated  31.8  calories  to  the  ice,  while 
25.4  calories  were  yielded  by  burning  enough  carbon  to  furnish  the 
amount  of  carbon  dioxide  exhaled  by  the  animal  in  the  same  period. 
The  figures  are  not  equal  and  we  now  know  why.  Apart  from  experi- 
mental errors  arising  from  the  unavoidably  imperfect  technic  of 
the  estimation,  the  animal  burnt,  not  only  carbon  during  the  period 
of  its  incarceration  in  the  ice-chamber,  but  also  hydrogen.  Were 
Carbohydrates,  in  which  the  hydrogen  is  fully  neutralized  by  oxygen 
already  present  in  the  molecule,  the  sole  source  of  energy,  then  the 
comparison  instituted  by  Lavoisier  would  have  been  adequate,  but  the 
Fats  and  Proteins  contain  an  excess  of  hydrogen,  of  which  the  heat  of 
combustion  must  be  added  to  that  of  the  carbon  in  order  to  establish 
the  chemical  origin  of  animal  heat  and  work.  Nevertheless  the  figures 
obtained  by  Lavoisier  were  sufficiently  comparable  to  afford  decided 
encouragement  to  the  view  which  he  himself  expressed :  "  La  vie  est 
une  fonction  chimique." 

In  1793  Lavoisier  was  condemned  to  death  and  executed  by  the 
apostles  of  Liberty,  Equality  and  Fraternity.  His  crime  appears  to 
have  consisted  in  his  being  a  man  of  superior  intellect  and  education 
who  had  dared  to  express  his  opinion  that  the  French  Academy  of 
Sciences  should  be  preserved  and  not  suppressed,  as  the  National 
Convention  desired.  His  appeal  for  liberty  to  live  and  serve  was  thus 
answered  by  the  president  of  the  tribunal  which  condemned  him: 


LAW  OF  THE  CONSERVATION  OF  ENERGY  569 

"La  Republique  n'a  pas  besoin  de  savants" — which  was  true,  until 
1870,  let  us  say,  or  1914.  It  is  to  a  Roman  politician  that  we  owe  the 
very  popular  and  oft-quoted  doctrine  that  "The  Voice  of  the  people 
is  the  Voice  of  God."  On  this  occasion  the  spokesman  of  the  people 
assured  one  of  the  greatest  discoverers  that  humanity  has  produced, 
that  a  republic  had  no  need  of  him  or  of  his  kind.  To  a  Swedish 
physicist,  Oersted,  we  owe  a  different  doctrine,  which  he  expressed  in 
these  words:  "The  Laws  of  Nature  are  the  Thoughts  of  God."  If 
we  should  estimate  the  value  of  these  two  doctrines  by  their  fruits, 
then  doubtless  we  would  prefer  the  doctrine  of  the  physicist  who 
produced  telegraphy  to  that  of  the  demagogue  who  planned  a  brutal 
and  senseless  murder.  Contemporary  events  will  doubtless,  in  time 
to  come,  furnish  us  with  an  abundance  of  additional  means  of  estimat- 
ing the  relative  value  of  these  theories. 

The  work  which  had  been  thus  initiated  by  Lavoisier,  was  carried 
on  by  his  pupil  Liebig,  who,  however,  mainly  devoted  his  attention  and 
his  life's  work  to  the  firm  establishment  of  the  Law  of  the  Conser- 
vation of  Matter  in  its  application  to  living  organisms.  The  methods 
of  organic  analysis  which  he  devised,  and  the  investigations  which  he 
undertook,  laid  the  foundations  of  analytical  biochemistry  as  we  know 
it  today.  The  energy-transformations  of  life  were  destined  to  become 
the  preoccupation  of  Liebig's  pupil,  Voit,  and  of  a  series  of  investi- 
gators who  owed  to  Voit  their  inspiration.  Thus,  to  the  second  and 
third  generations  of  investigators  succeeding  Lavoisier,  fell  the  task 
of  achieving  the  fruition  of  his  labors. 

In  order  to  render  possible  an  accurate  comparison  of  the  kind  which 
was  attempted  by  Lavoisier  it  was  first  of  all  necessary  to  ascertain 
Heats  of  Combustion  of  the  various  foodstuffs.  The  actual  fuels  burnt 
by  the  animal  machine  are  carbohydrates,  fats  and  proteins,  and  it 
is  evidently  with  the  heat  of  combustion  of  these  substances,  and  not 
merely  that  of  carbon,  tjiat  we  should  compare  the  heat-evolution  of 
an  animal. 

The  Calorific  Values  in  heat-units  per  gram  for  the  different  repre- 
sentatives of  the  three  main  classes  of  foodstuffs  do  not  vary  greatly 
among  themselves.  The  molecules  of  the  Fats  and  Proteins  are 
so  large  that  the  differences  of  composition  or  structure  which  they 
display  affect  the  total  heat  of  combustion  but  slightly,  while  the 
Carbohydrates  uniformly  contain  the  proportion  of  oxygen  which  is 
requisite  to  burn  their  hydrogen  and  hence  the  combustion-value  for 
each  carbohydrate  is  very  nearly  proportional  to  the  carbon  which  it 
contains  and  this  in  turn  is  proportional  to  the  weight  of  the  molecule. 
The  following  are  the  calorific  values  of  various  foodstuffs,  as  esti- 
mated by  complete  combustion  in  a  calorimeter,  the  heat-output 
being  expressed  in  terms  of  the  large  calorie,  or  quantity  of  heat  re- 
quired to  raise  the  temperature  of  one  kilogram  of  water  from  0°  C. 
to  1°  C. 


570  THE  ANIMAL  BODY  AS  A  MACHINE 

Cals. 
Proteins: 

Casein       .      .      .      .........    ....      r    .      .     5.86 

Egg-albumin 5.74 

Serum-albumin 5.92 

Average 5.84 

Fats: 

Tissue-fat .  9 . 48 

Butter-fat - 9.23 

Olive  oil 9.33 

Average 9.35 

Carbohydrates : 

Glucose 3.74 

Cane-sugar 3.96 

Milk-sugar .  3.95 

Maltose 3.95 

Starch 4.18 

Average 3.96 

The  figures  usually  employed  for  the  fats  and  carbohydrates  as 
they  actually  occur  in  a  mixed  diet  are  those  which  were  originally 
estimated  by  Rubner,  namely: 

One  gram  of  fat  =  9.3  calories 

One  gram  of  carbohydrate  =  4.1  calories 

the  high  value  for  carbohydrates  being  employed  on  account  of  the 
predominance  of  starch  among  the  carbohydrates  of  an  ordinary 
mixed  diet. 

The  heat-value  of  carbohydrates  and  fats  for  the  body  must  be  the 
same  as  that  indicated  by  the  combustion-calorimeter,  since  the 
products  of  combustion  are  in  both  cases  identical,  namely,  carbon 
dioxide  and  water.  The  case  is  far  different  for  the  Proteins,  however, 
because  these  are  not  completely  burnt,  the  nitrogen  being  excreted 
in  the  form  of  urea,  creatinine  and  so  forth,  which  are  substances  still 
capable  of  yielding  heat  when  they  are  completely  oxidized.  Further- 
more, the  proteins  as  they  actually  occur  in  the  diet  are  not  com- 
pletely digested  and  assimilated,  a  proportion  of  indigestible  or  diffi- 
cultly assimilable  material  being  evacuated  in  the  feces.  The  true 
heat-value  of  protein  to  the  animal  body  is  therefore  not  indicated  by 
the  combustion-calorimeter. 

The  determination  of  the  actual  calorific  value  of  protein  in  the 
animal  body  was  first  carried  out  by  Rubner.  His  procedure  was  as 
follows:  The  calorific  value  of  dried  muscle-tissue  was  determined 
in  the  combustion-calorimeter,  and  the  heat- values  of  the  urine  and 
feces  upon  an  exclusive  meat-diet  were  also  determined.  Subtract- 
ing the  heat-value  of  the  excreta  from  that  of  the  food,  and  also  a 
small  correction  representing  the  heat  of  solution  of  the  urea  in  the 
urine,  it  was  found  that  an  average  of  about  4.1  calories  per  gram 
was  actually  available  to.  the  animal  from  the  protein  in  its  diet.  The 


LAW  Of  THE  CONSERVATION  OF  ENERGY  571 

actual  calorific  value  of  a  protein  to  an  animal  is  therefore  the  same  as 
that  of  a  carbohydrate,  both  being  far  inferior  in  heat-value  to  the 
fats. 

The  necessary  data  for  the  accurate  evaluation  of  the  comparison 
which  Lavoisier  attempted  were  by  now  assembled  and  the  com- 
parison, when  actually  carried  out  by  Rubner  in  1894,  established 
beyond  any  doubt  the  validity  of  the  principle  of  the  Conservation  of 
Energy  in  the  phenomena  of  life.  The  experiments  were  carried  out 
upon  a  dog,  because  there  existed  at  that  time  no  calorimeter,  of 
sufficient  size  to  contain  a  man,  which  would  accurately  measure  the 
heat  evolved  during  a  period  of  twenty-four  hours.  The  heat  actually 
imparted  by  the  dog  to  the  calorimeter  in  twenty-four-hour  periods 
was  measured  and  this  was  compared  with  the  heat-value  of  its  food 
computed  from  the  nitrogen  in  the  urine  (1  gram  Nitrogen  =  6.25 
grams  protein  =  25.63  calories)  and  from  the  output  of  water  and 
carbon  dioxide.  The  following  are  the  details  of  his  comparisons,  the 
"food"  in  starvation  consisting,  of  course,  of  the  proteins  and  fats  of 
the  animal's  own  tissues: 

Number  of  Heat  calculated          Heat  directly  Difference  in 

Food.  days.  from  metabolism.  determined.  percentage. 

-1.42 


2 

Fat     .      .    \      .     5  1510.1  1498.3  -0.97 

2488.0 


Meat  and  fat 


/     '      '      '      6  2249'8  2276.91 

\     .      .      .      7  4780.8  4769.3  / 


iiyr  A      I  V  Xf^rtJJ.O  ^~t\J.V       I  0.42 

iviGo/t  \  „  n-ic\n  c\  Ant*f\  *\    t         ,  _i_n  j.^? 


When  one  considers  the  complexity  of  these  estimations,  the  multi- 
tude of  factors  which  participate  in  determining  their  outcome,  and 
the  elaborate  character  of  the  apparatus  employed,  the  coincidence 
of  the  calculated  and  actual  output  is  so  exact  as  to  leave  no  room  for 
doubt  that  the  law  of  the  conservation  of  energy  applies  no  less  to 
animals  than  to  other  machines.  The  energy  which  the  animal  dis- 
sipates is  derived  from  the  combustion  of  foodstuffs,  just  as  the  energy 
dissipated  by  a  locomotive  is  derived  from  the  oxidation  of  its  fuel. 
In  the  living,  as  in  the  inanimate  machine,  the  potential  energy  of  the 
fuel  is  released  by  oxidation  and  reappears  in  the  form  of  heat  and 
work. 

An  even  more  exact  balance  between  income  and  output  was  how- 
ever sought  for  and  found  by  the  American  investigator,  Atwater. 
The  extraordinary  degree  of  accuracy  which  was  attained  in  his 
investigations  was  rendered  possible  by  the  invention  of  the  Atwater- 
Rosa  Calorimeter,  which  was  of  sufficient  capacity  to  hold  a  man  and 
yet  so  technically  perfect  that  when  a  measured  amount  of  heat  was 
generated  within  the  calorimeter  by  an  electric  current,  the  quantity 
of  heat  liberated  could  be  measured  to  within  0.01  per  cent.  (Figs.  48 
and  49).  The  amount  of  protein  burnt  by  the  subject  was  estimated 


572 


THE  ANIMAL  BODY  AS  A   MACHINE 


from  the  nitrogen  in  the  urine  and  in  the  feces.  The  carbon  which 
would  be  derived  from  this  quantity  of  protein  was  deducted  from  the 
total  carbon  output  and  the  difference  yielded  the  total  non-protein 
carbon,  or  carbon  derived  from  carbohydrates  and  fat.  The  carbo- 
hydrates in  the  food  were  measured  and  the  corresponding  quantity 
of  carbon  deducted  from  the  total  non-protein  carbon.  The  difference 
represented  carbon  derived  from  the  fat.  In  this  way  the  quantities 
of  each  of  the  three  classes  of  foodstuffs  consumed  were  estimated  and 


FIG.  48. — Schematic  diagram  of  the  Atwater-Rosa-Benedict  respiration-calorimeter. 
02,  oxygen  introduced  as  consumed  by  subject;  3,  H2SQ4  to  catch  moisture  given  off  by 
soda-lime;  2,  soda-lime  to  remove  COz',  1,  H2SO4  to  remove  moisture  given  off  by  subject; 
Bl,  blower  to  keep  air  in  circulation;  V,  vacuum  jacket;  C,  tank  for  weighing  water 
which  has  passed  through  calorimeter  each  hour;  W,  thermometer  for  measuring  tem- 
perature of  wall;  Ai,  thermometer  for  measuring  temperature  of  the  air;  R,  rectal  ther- 
mometer for  measuring  temperature  of  subject.  (After  Lusk.) 

the  energy  which  their  combustion  could  yield  was  computed  in  the 
manner  indicated  above  and  compared  with  the  actual  heat-evolution 
of  the  subject.  The  results  of  forty  days'  experimentation  with  three 
different  subjects  yielded  the  following  averages: 

Calculated  daily  output .      .      .     «.      .      .      ,/,•.,      .  '  .      .      2717  calories 

Observed  daily  output 2723        " 

Difference,  0.2  per  cent. 

A  further  refinement  of  technic  consisted  in  the  simultaneous  esti- 
mation of  the  carbon-dioxide  output  and  the  oxygen  intake,  from 


LAW  OF  THE  CONSERVATION  OF  ENERGY 


573 


which  the  Respiratory  Quotient  could  be  calculated.  Deducting  the 
protein  carbon  from  the  total  carbon  output,  and  the  oxygen  required 
to  oxidize  the  protein  from  the  total  oxygen  intake,  the  ratio  of  the 
non-protein  carbon  dioxide  to  the  residual  oxygen  intake,  or  the  non- 
protein  respiratory  quotient;  afforded  a  measure  of  the  proportion  of 
fat  to  carbohydrate  actually  consumed  by  the  subject  of  the  experi- 
ment. Thus  a  non-protein  respiratory  quotient  of  0.707  indicates  the 


FIG.  49. — General  view  of  the  respiration-calorimeter  laboratory  at  Middletown, 
Connecticut.  The  calorimeter-chamber  is  seen,  with  window  open  upon  the  right. 
The  principle  of  its  construction  is  that  of  an  ordinary  refrigerator,  namely,  a  chamber 
surrounded  by  a  series  of  confined  air-spaces.  The  inner  chamber  is  of  copper.  This  is 
succeeded  by  a  wall  of  zinc  and  two  walls  of  wood,  each  pair  of  walls  being  separated  by 
about  three  inches  of  air-space.  Gain  or  loss  of  heat  through  the  metallic  walls  of  the 
chamber  is  prevented  by  keeping  the  zinc  wall  at  the  sa.me  temperature  as  the  copper. 
Any  difference  of  temperature  between  these  two  walls  is  indicated  by  a  thermocouple 
and  a  galvanometer.  Heat  is  supplied  to  the  air-space  surrounding  the  zinc  wall  by 
passing  an  electrical  current  through  coils  of  resistance-wire.  Cooling  is  accomplished 
by  currents  of  water.  The  heat  generated  in  the  chamber  is  removed  partly  in  the 
form  of  the  latent  heat  of  vaporization  of  the  water  exhaled  from  the  lungs  and  partly 
by  means  of  cold-water  absorbers.  The  quantity  of  heat  evolved  is  computed  from  the 
amount  of  water  passing  through  the  heat-absorbers  and  its  rise  in  temperature  during 
its  passage.  (After  Benedict  and  Milner.) 

combustion  of  pure  fat,  a  quotient  of  1 .00  indicates  the  combustion  of 
pure  carbohydrate  (cf .  Chapter  XXII)  and  intermediate  values  repre- 
sent the  combustion  of  a  mixture  of  these  foodstuffs,  the  composition 
of  which  can  be  estimated  by  a  simple  calculation. 

It  now  remained,  in  order  to  complete  the  demonstration  of  the 
validity  of  the  law  of  the  conservation  of  energy  in  the  animate  world, 
to  investigate  the  source  of  the  energy  which  is  expended  by  an  animal 
in  the  performance  of  external  work.  In  the  experiments  hitherto 


574  THE  ANIMAL  BODY  AS  A   MACHINE 

enumerated  the  subjects  were  at  rest,  and  although  their  respiratory 
and  cardiac  muscles  were  contracting  and  the  skeletal  muscles  main- 
tained in  tone  or  even  contracting,  yet,  the  whole  of  the  organism  being 
enclosed  within  a  heat-insulated  system,  the  effect  of  all  these  move- 
ments ultimately  appeared  and  was  estimated  in  the  form  of  heat. 
The  case  is  different  when,  as  in  many  of  Atwater's  experiments,  the 
subject  was  made  to  perform  external  work,  by  operating  a  stationary 
bicycle  which  was  so  arranged  that  the  rotation  of  the  wheels  raised 
a  weight.  The  energy  output  was  not  in  this  case  expressed  entirely 
in  the  form  of  heat,  but  in  part  in  the  form  of  Mechanical  Work.  We 
can  express  this  work  in  terms  of  heat-units,  however,  just  as  we  can 
express  heat  in  terms  of  electrical  units  or  electrical  units  in  terms  of 
mechanical  work  again.  Since  no  energy  is  ever  lost  and  all  forms  of 
energy  are  equivalent  to  one  another,  the  heat-value  consumed  in 
performing  mechanical  work  can  be  directly  calculated  from  the  known 
mechanical  equivalent  of  heat.  The  following  are  the  results  which 
Atwater  obtained  in  the  investigation  of  this  problem: 

Calories. 


Income  per  Output  per 

twenty-four  twenty-four  Difference. 

Days.                        hours.  hours.  per  cent. 
Rest  experiments: 

7  experiments  with  E.G.  .      25                     2268  2259  -0.4 

1  experiment  with  A.W.S.       3                     2304  2279  -1.1 

3  experiments  with  J.F.S.       9                     2118  2136  +0.8 

1  experiment  with  J.C.W.       4                     2357  2397  +1.7 


Average     ."     .      ,  41  2246  2246  ±0.0 

Work  experiments : 

2  experiments  with  E.G.    .  8  3865  3829  -0.9 

4  experiments  with  J.F.S.  12  3539  3540  ±0.0 

14  experiments  with  J.C.W.  46  5120  5120  ±0.0 


Average     ...     66  4682  4676  -0.1 

To  within  one  part  in  a  thousand  the  output  of  heat  plus  work  was 
equal  to  the  calorific  value  of  the  foodstuffs  consumed .  We  can  hardly 
doubt  that  this  minute  discrepancy  was  of  purely  technical  origin  and 
that  these  experiments  represent  the  culmination  of  the  proof  which 
Lavoisier  had  sought  a  hundred  years  previously,  that  the  energies  of 
life  are  derived  simply  and  solely  from  the  chemical  energy  of  the 
foodstuffs. 

The  fundamental  importance  of  these  investigations  cannot  be  over- 
rated, for  they  reveal  to  us  in  the  clearest  possible  manner  the  fact  that 
life  is  the  outcome  of  a  complex  of  forces  which  it  does  not  create.  We 
are  enabled  by  them  to  confidently  state  that  if  there  is  such  an  entity 
as  "Vital  Force"  created  and  generated  out  of  nothing  by  living  organ- 
isms, then  the  inconspicuousness  of  its  effects  is  commensurate  with  the 
inconspicuousness  of  its  origin.  They  must  be  confined  to  somewhat 
Jess  than  a  one  thousandth  part  of  the  total  activity  of  the  organism. 


ISODYNAMIC   VALUES  OF  THE  FOODSTUFFS  575 

We  must  be  careful,  however,  in  formulating  any  such  fundamental 
conclusion  not  to  go  too  far.  We  must  beware  of  overstepping  to  the 
slightest  extent  the  sure  ground  of  fact  which  our  evidence  affords,  and 
we  must  therefore  candidly  admit  that  while  the  evidence  accumulated 
by  the  remarkable  series  of  investigations  which  we  have  briefly  and 
inadequately  outlined,  clearly  justifies  the  conclusion  that  no  source  of 
energy  is  contributed  to  or  resides  in  the  organism  that  is  not  com- 
prised in  the  chemical  energy  of  its  foodstuffs,  and  the  heat  of  its 
environment,  yet  we  cannot  definitely  reject  the  possibility  that  forces 
of  evanescent  magnitude  which  are  not  comprised  in  either  of  the 
above  categories  may  influence,  in  the  manner  of  a  catalyzer,  the  rate  of 
discharge  of  energy  from  the  organism.  We  cannot  disprove  this,  but 
then,  on  the  other  hand,  if  one  should  choose  to  assume  the  existence 
of  such  forces  the  burden  of  proof  clearly  rests  upon  the  originator  of 
the  hypothesis.  In  the  interpretation  of  life-phenomena,  so  far  as  we 
have  as  yet  been  enabled  to  subject  them  to  measurement,  such  an 
assumption  has  proved  to  be  altogether  unnecessary,  and  hence  our 
present  state  of  knowledge  affords  for  it  no  foundation  whatever. 
No  sure  ground  is  possible  in  scientific  discovery  unless  we  proceed  from 
the  known  to  the  unknown.  The  assumption  that  hitherto  unknown 
forces  are  involved  in  life  cannot  assist  but  only  retard  its  interpreta- 
tion until  and  unless  every  previously  known  possibility  has  been 
exhausted  in  a  vain  endeavor  to  reconcile  the  facts.  But  the  existence 
of  unknown  possibilities  manifestly  cannot  be  contradicted  upon  a 
priori  grounds,  and  a  dogmatic  insistence  upon  the  sufficiency  of  the 
known  has  only  too  frequently,  in  the  history  of  science,  served  but  to 
pave  the  way  for  a  subsequent  recantation. 

THE  ISODYNAMIC  VALUES  OF  THE  FOODSTUFFS. 

Since  the  products  of  the  combustion  of  the  Fats  and  Carbohydrates 
in  the  diet  are  the  same,  namely  carbon  dioxide  and  water,  it  was  sug- 
gested at  an  early  period  in  the  investigation  of  metabolism  that  these 
components  of  the  dietary  might  be  mutually  interchangeable  in 
equicalorific  quantities.  This  possibility  was  experimentally  realized 
by  Rubner,  who  fpund  that  100  grams  of  fat  in  the  diet  could  be 
replaced  by  232  grams  of  starch  or  234  grams  of  cane-sugar,  the  equi- 
calorific values  estimated  from  the  heat  of  combustion  being  229  grams 
of  starch  and  235  grams  of  cane-sugar.  The  same  conclusion  was  ulti- 
mately reached  by  Atwater  in  a  series  of  experiments  in  which  the 
subjects  were  made  to  perform  external  work,  so  that  part  of  the  energy 
of  the  foodstuffs  had  to  be  expended  for  this  purpose.  The  procedure 
of  the  experiments  was  designed  to  test  the  efficacy  of  the  fats  as  sub- 
stitutes for  carbohydrates  in  a  variety  of  ways.  Thus  the  diet  was 
insufficient  to  maintain  bodily  equilibrium,  so  that  there  was  a  loss  of 
weight  throughout  the  duration  of  the  experiments  due  to  the  consump- 
tion of  the  subject's  tissues.  The  loss  of  body-substance  on  the  diet 


576 


THE  ANIMAL  BODY  AS  A  MACHINE 


containing  carbohydrates  could  thus  be  compared  with  that  experienced 
on  the  diet  containing  fats,  and  the  relative  value  of  these  constituents 
of  the  dietary  as  tissue-sparers  could  thus  be  estimated.  The  external 
Mechanical  Work  performed  in  both  sets  of  experiments  was  as  nearly 
as  possible  the  same,  and  equivalence  of  total  energy-consumption  on 
the  two  diets  would  therefore  indicate  equal  availability  of  fats  and  of 
carbohydrates  for  the  performance  of  mechanical  work.  The  following 
table  summarizes  .the  results  of  these  experiments: 


Experiment  number. 

Time, 
days. 

Heat 
derivable 
from  food, 
calories. 

Heat 
equivalent 
of  external 
work, 
calories. 

Total 
energy- 
output, 
calories. 

Calories 
equivalent 
to  gain  (  +) 
or  loss  (  -) 
of  tissue. 

40    J.C.W.  carbohydrate-diet     . 

4 

4180 

518 

5251 

-1071 

41     J.C.W.  fat-diet     .... 

4 

4150 

522 

5304 

-1154 

44     J.C.W.  carbohydrate-diet     . 

4 

4602 

571 

5125 

-523 

43     J.C.W.  fat-diet     .... 

4 

4496 

548 

5155 

-659 

47     J.C.W.  carbohydrate-diet      . 

4 

4366 

562 

5173 

-807 

46     J.C.W.  fat-diet      .... 

4 

4%473 

551 

5193 

-715 

53     J.C.W.  carbohydrate-diet     . 

3 

5132 

587 

5104 

+28 

52     J.C.W.  fat-diet     .... 

3 

5120 

607 

5309 

-189 

Average  of  four  experiments  with 

carbohydrate-diet    .... 

15 

4532 

558 

5167 

-635 

Average  of  four  experiments  with 

fat-diet 

15 

4524 

554 

5236 

—  712 

The  substantial  equivalence  of  the  fats  and  carbohydrates  as  sources 
of  heat  and  work  and  sparers  of  tissue  in  these  experiments  is  evident. 
There  is  some  indication  that  the  loss  of  tissue  on  the  fat-diet  is  greater 
than  it  is  on  a  carbohydrate-diet,  and  this  is  especially  evident  in  the 
experiment  in  which  the  total  calorific  value  of  the  diet  was  relatively 
high.  The  reason  for  this  probably  lies  in  the  fact  that,  as  Zeller  has 
recently  shown,  if  the  preponderance  of  fat  over  carbohydrates  in  the 
diet  be  too  great,  even  when  the  total  calorific  value  of  the  diet  is  kept 
constant,  acetone  bodies  appear  in  the  urine  and  an  Acidosis  arises 
necessitating  the  production  of  Ammonia  by  the  tissues  tc  neutralize 
the  excess  of  acid  radicals  in  the  blood.  The  output  of  nitrogen  is 
consequently  increased  and  loss  of  body-substance  accelerated.  This 
effect  only  appears  in  normal  individuals,  however,  when  less  than  ten 
per  cent,  of  the  total  calories  are  given  in  the  form  of  carbohydrate. 
Up  to  this  limit,  therefore,  the  carbohydrates  in  the  diet  may  be  replaced 
by  fat  without  influencing  very  appreciably  the  total  heat-output  or 
wastage  of  tissue-materials.  In  Diabetes,  of  course,  the  limit  of  toler- 
ance for  fats  is  much  lower  than  this. 

In  the  replacement  of  the  fats  by  carbohydrates  we  are  limited  in 
another  direction.  So  far  as  the  mere  question  of  heat-equivalence 
is  concerned  the  complete  replacement  of  the  fats  in  the  dietary  by 
carbohydrates  is  doubtless  entirely  feasible,  more  especially  since  the 
conversion  of  carbohydrates  into  body-fat  is  a  regular  concomitant  of 


ISODYNAMIC   VALUES  OF  THE  FOODSTUFFS  577 

insufficient  utilization  of  the  carbohydrates  of  the  diet  for  the  produc- 
tion of  heat  and  work.  We  have  seen  (Chapter  XX),  however,  that 
certain  essential  Substrates  of  Growth,  or  raw  materials  for  the  synthesis 
of  protoplasm  are  contained  in  the  animal  fats  and,  so  far  as  we  are 
yet  aware,  in  no  other  abundant  constituents  of  the  diet.  The  total 
replacement  of  fats  by  carbohydrates,  therefore,  is  likely  to  result  in 
unbalanced  tissue-waste  through  the  lack  of  non-synthesizable  atom- 
complexes  which  do  not  necessarily  contribute  any  appreciable  share  to 
the  energy-output.  The  total  replacement  of  animal  fats  by  Vegetable 
Oils  is  for  a  like  reason  impracticable.  The  proportion  of  animal  fat 
which  is  requisite  for  maintenance  is,  however,  very  small,  and  pro- 
vided this  small  residuum  is  retained,  the  fats  of  the  dietary  may  be 
replaced  by  carbohydrates  in  equicalorific  proportions  without  affect- 
ing the  balance  of  energy-input  and  -output. 

In  the  case  of  the  Proteins  a  number  of  complications  arise  which 
limit  in  a  variety  of  directions  the  application  of  the  principle  of 
isodynamic  values.  In  the  first  place  the  proteins  are  the  medium 
through  which  the  body  acquires  its  nitrogen.  Their  complete  replace- 
ment by  fats  or  carbohydrates  is  therefore  obviously  impossible.  Then, 
again,  different  types  of  protein  are  not  even  isodynamic  with  each 
other,  for  those  which  lack  or  are  deficient  in  certain  amino-acids,  such 
as  Gelatin,  Zein  or  Gliadin  will  not  replace  the  protein  in  a  normal 
mixed  diet  however  great  an  excess  of  the  incomplete  protein  may 
.be  employed  (Chapter  XX).  No  nitrogen  balance  is  possible  unless 
the  missing  amino-acids  are  supplied,  and  upon  a  diet  containing  an 
abundance  of  nitrogen  the  output  will  continuously  exceed  the  intake. 
If,  however,  the  missing  amino-acids  are  added  to  these  proteins,  as, 
for  example,  tyrosine,  cystine  and  tryptophane  to  gelatin,  then  the 
attainment  of  nitrogenous  equilibrium  becomes  possible  because  all  of 
the  constituent  parts  of  tissue-protein  are  then  present  in  the  diet. 

Although  gelatin  cannot  replace  other  proteins  in  the  diet,  yet  it  is 
possible  to  attain  nitrogenous  equilibrium  on  a  smaller  amount  of 
normal  dietary  protein  if  gelatin  be  also  present.  If  the  total  heat- 
requirement  of  the  normally  fed  animal  be  supplied  solely  in  the  form  of 
carbohydrates  and  fats  a  certain  daily  loss  of  nitrogen  will  occur  which 
is  due  to  the  consumption  of  tissue-proteins.  If  7.5  per  cent,  of  the 
heat- value  be  now  supplied  in  the  form  of  gelatin  the  excess  of  loss  over 
intake  is  diminished  by  23  per  cent.  If,  however,  60  per  cent,  of  the 
heat-value  of  the  food  is  supplied  by  gelatin  the  saving  of  tissue-protein 
is  only  35  per  cent.,  and  if  the  whole  of  the  heat-value  be  supplied 
in  gelatin  only  37.5  per  cent,  of  the  tissue-wastage  is  spared.  The 
principle  of  isodynamic  values  is  therefore  manifestly  inapplicable  to 
the  quantitative  relationship  between  gelatin  and  the  other  dietary 
constituents  unless  a  sufficiency  of  other  protein  be  at  the  same 
time  supplied  to  furnish  the  full  requirement  of  tyrosine,  cystine,  and 
tryptophane. 

A  further  limitation  upon  the  application  of  the  principle  of  isody- 
37 


578  THE  ANIMAL  BODY  AS  A   MACHINE 

namic  values  to  the  protein  constituents  of  the  dietary,  arises  from  the 
fact  that  an  increase  of  protein  in  diet  actually  stimulates  the  total 
metabolism,  so  that  more  food  is  burnt  and  more  heat  evolved  on  a  diet 
high  in  protein  than  upon  a  diet  which  contains  less  protein.  This 
phenomenon,  which  Rubner  terms  the  Specific  Dynamic  Action  of  pro- 
teins, is  very  well  displayed  by  the  effect  of  administering  protein  to  a 
starving  animal.  One  might  suppose  that  if  a  starving  animal  is  losing 
a  certain  amount  of  tissue-protein  daily,  the  administration  of  this 
amount  of  protein  daily  would  suffice  to  balance  the  nitrogenous  input 
and  output.  This  is  not  the  case,  however,  for  on  increasing  the 
nitrogenous  input  an  increase  of  nitrogenous  output  also  occurs  and 
the  balance  remains  negative.  A  further  increase  of  nitrogenous  input 
calls  forth  a  still  greater  metabolism  of  protein  until,  on  an  exclusively 
protein  diet,  a  balance  between  intake  and  output  is  attained  with  an 
output  of  nitrogen  no  less  than  three  and  one-half  times  that  which  is 
observed  in  the  starving  animal.  In  man  the  quantity  of  protein  thus 
required  to  obtain  nitrogenous  equilibrium  is  greater  than  he  can 
conveniently  consume,  and  even  when  nitrogenous  equilibrium  has 
been  attained  the  carbon  balance  remains  negative,  since  not  only  the 
nitrogenous  metabolism,  but  the  metabolism  of  fats  and  carbohydrates 
is  stimulated  by  protein.  The  effect  of  protein  is  therefore  to  greatly 
increase  the  heat-evolution  of  the  body,  and  the  replacement  of  fat  or 
carbohydrate  by  protein  in  a  diet  which  is  just  sufficient  to  maintain 
equilibrium  results  in  rendering  the  diet  inadequate  to  replenish  the 
tissue-loss.  The  proteins  cannot,  therefore,  replace  fats  or  carbo- 
hydrates in  isodynamic  proportions. 

The  origin  of  the  specific  dynamic  action  of  the  proteins  has  been 
sought  by  Lusk,  who  investigated  the  effects  of  individual  Amino- 
acids  upon  the  heat-output  in  starving  dogs.  He  found  that  while 
glycocoll  and  alanine  greatly  increase  the  production  of  heat,  and 
leucine  and  tyrosine  slightly,  glutamic  acid  is  devoid  of  action.  A 
mixture  of  5.5  grams  each  of  glycocoll,  alanine,  glutamic  acid  and 
tyrosine  produced  as  much  increase  of  heat-output  as  100  grams  of 
meat. 

THE  PROTEIN  REQUIREMENT  IN  THE  DIETARY. 

From  the  preceding  considerations  it  must  be  evident  that  the 
proteins  are  the  most  wasteful  constituents  of  the  dietary,  since  they 
increase  the  consumption  of  other  constituents  as  well  as  that  of  pro- 
tein itself.  The  proteins  are  also  the  most  expensive  foodstuffs  from 
a  commercial  point  of  view  and  this  is  particularly  true  of  the  proteins 
of  animal  origin,  for  while  there  is  little  wastage  of  energy  or  materials 
in  the  growth  of  the  vegetable  constituents  of  the  diet,  a  very  large 
wastage  occurs  in  the  synthesis  of  animal  proteins  for  human  consump- 
tion. An  ox  or  sheep  may,  for  our  immediate  purpose,  be  regarded  as 
an  ambulatory  factory  of  protein.  In  order  to  supply  this  factory  with 
raw  materials,  vegetable  proteins,  carbohydrates  and  fats  must  first 


PROTEIN  REQUIREMENT  IN  THE  DIETARY  579 

be  grown  at  the  expense  of  the  constituents  of  the  soil  and  the  pre- 
occupation of  space  that  might  be  otherwise  utilized.  Not  only  must 
an  amount  of  vegetable  food  be  provided  equivalent  in  heat-value  to 
the  animal  foodstuffs  which  we  desire  to  synthesize,  but  an  enormous 
excess,  to  supply  the  radiation  of  heat  and  mechanical  work  performed 
by  the  animal  throughout  the  period  of  its  growth.  The  Animal 
Proteins  therefore  represent  a  consumption  and  expenditure  of  food- 
materials  totally  disproportionate  to  their  calorific  value.  The 
vegetable  proteins,  on  the  other  hand,  are  also  expensive  because  the 
proportion  of  protein  in  the  majority  of  vegetable  tissues,  with  few 
exceptions,  is  extremely  small. 

As  a  measure  of  national  economy,  therefore,  if  we  view  the  matter 
solely  from  a  financial  standpoint,  a  restriction  of  the  protein-con- 
sumption to  the  minimum  consistent  with  health  and  efficiency  would 
seem  to  be  highly  desirable.  Now  the  consumption  of  protein  food- 
stuffs and  particularly  of  animal  proteins  varies  very  greatly  among 
different  peoples.  The  following  pre-war  statistics  are  furnished  by 
Ostertag : 

Meat  consumed 

per  day  per 

capita, in 

grams. 

Australia .      ....  >\  *  •..'..      .  306 

United  States  of  Ameri  ca 149 

Great  Britain     .....      ,      .      .     „      .      .      .      ...      .      .  130 

France .      .  -..      .*%      .....      .      .  92 

Belgium  and  Holland   . • ...      .      .      .    :.      .      .      .  86 

Austria-Hungary 79 

Russia     ....."...    ",'•   .      .      .      .      .      .-•    .      .•.'  -.  59 

Spain 61 

Italy.      .      ,    ,V  -.      ...A    ...;•.   -.  ...; ":.-.,  :..     .......  29 

Japan 25 

It  will  be  observed  that  the  consumption  of  meat  in  the  English- 
speaking  countries  far  exceeds  that  which  prevails  elsewhere.  Either 
the  English-speaking  countries  and  particularly  Australia  are  waste- 
fully  dissipating  their  food-values,  or  else  a  large  proportion  of  the 
population  of  Europe  is  chronically  suffering  from  suboptimal  con- 
sumption of  protein. 

The  standard  requirement  of  protein,  partially  derived  from  meat 
and  in  part  from  vegetables  and  cereals,  was  computed  by  Voit  to 
be  118  grams  for  the  average  man  not  engaged  in  heavy  labor,  and 
90  grams  for  a  woman.  This  estimate  was  based  upon  a  statistical 
comparison  of  the  actual  consumption  by  presumably  normal  persons 
subsisting  upon  a  mixed  diet.  The  necessity  for  this  intake  of  protein 
has  of  recent  years,  however,  been  sharply  challenged  by  Chittenden 
and  others  of  the  American  school  of  physiologists  and  biological 
chemists.  The  statistical  method  of  estimating  protein-requirements 
is  based  upon  the  assumption  of  the  exercise  of  free  choice  by  the 
individual  and  the  underlying  supposition  is  made  that  prevailing  diets 
represent  a  species  of  "  survival  of  the  fittest/'  It  is  obvious,  however, 


580  THE  ANIMAL  BODY  AS  A   MACHINE 

that  if  this  criterion  were  to  be  applied  in  Japan  it  would  yield  far 
different  estimates  from  those  which  would  result  from  its  application 
in  England.  As  Taylor  has  observed,  the  customary  dietary  of  dif- 
ferent races  has  in  no  small  degree  been  fashioned  by  their  ethnological 
development.  "In  some  lands  races  were  compelled  to  adopt  cultiva- 
tion of  the  soil,  in  other  places,  fishing,  in  some  areas  the  chase  remained, 
long  into  relative  civilization,  one  of  the  chief  methods  of  securing  food. 
The  variations  in  ethnological  development  brought  about  by  enforced 
cultivation  of  the  soil,  as  contrasted  with  the  state  of  affairs  in  a  tribe 
of  hunters,  are  well  illustrated  in  different  tribes  of  our  American 
Indians.  Depending  upon  the  method  of  sustaining  the  life  of  the 
tribe,  the  standard  diet  of  the  tribe  varied.  Only  under  modern 
conditions  of  transportation  have  the  instincts  and  tastes  of  man  had 
opportunity  for  full  choice  in  diet.  Compulsion  to  some  extent  and  in 
some  degree  there  has  always  been." 

Chittenden  was  able  not  only  to  maintain  nitrogenous  and  calorific 
equilibrium  for  prolonged  periods  on  a  much  lower  protein  intake 
than  that  recommended  by  Voit,  but  he  was  able  to  keep  athletes  in  a 
condition  fitting  them  for  extreme  exertion.  According  to  Taylor  the 
Nitrogenous  Metabolism  of  a  man  of  70  kilos  may  be  summarized  as 
follows,  the  nitrogenous  output  being  expressed  in  terms  of  grams  of 
protein : 

Grams  per 
day. 

Nitrogen  output  on  protein-free  diet  with  carbohydrates        .      .      .  10  to   15 

Nitrogen  output  in  starvation,  lowest  level 15  to  20 

Nitrogenous  and  caloric  equilibrium,  with  ample  ingestion  of  carbo- 
hydrate       30 

Nitrogenous  and  caloric  equilibrium,  largely  with  fat 40 

Normal  protein  input,  safety  margin  of  100  per  cent 70 

Nitrogenous  and  caloric  equilibrium  on  a  pure  protein  diet    .      .      .  750 

Nitrogenous  and  calorific  equilibrium  can  therefore  be  attained  on  a 
diet  rich  in  carbohydrates  with  a  daily  intake  of  only  one-third  of  the 
amount  of  protein  recommended  by  Voit.  It  cannot  be  positively 
affirmed  that  this  low  protein  intake  would  also  suffice  to  permit 
normal  growth  in  children  or  adolescents.  It  has  been  argued  that  as 
a  great  part  of  even  this  small  protein  intake  is  simply  deaminized 
and  burnt  in  the  Exogenous  Metabolism  there  must  be  plenty  to  spare 
for  tissue-synthesis.  It  has  never  been  demonstrated,  however,  that 
the  exogenous  metabolism  is  reducible  below  a  certain  level.  In  fact 
the  deaminization  of  amino-acids  with  production  of  urea  continues 
even  in  starvation.  There  is  apparently,  in  so  far  as  protein  is  con- 
cerned, no  level  of  the  nutrient-reservoir  at  which  a  large  overflow  does 
not  occur.  If  the  overflow  and  inflow  are  nearly  balanced  and  the  over- 
flow (i.  e.,  exogenous  metabolism)  is  irreducible  upon  a  diet  of  given 
composition,  then  it  is  clear  that  the  outflow  of  nutrients  to  the  tissues 
may  be  just  sufficient  to  maintain  repair  and  yet  quite  inadequate  to 
synthesize  additional  tissue,  despite  the  fact  that  the  intake  is  far  above 


PROTEIN  REQUIREMENT  IN  THE  DIETARY  581 

that  which  would,  in  the  absence  of  the  overflow,  be  necessary  for  this 
purpose.  In  order  to  establish  the  adequacy  of  a  maintenance-income 
of  protein  for  growth  it  would  be  necessary  to  show  that  the  rate  of 
exogenous  metabolism,  which  appears  to  be  governed,  at  least  in  part, 
by  the  Thyroid,  is  reduced  when  tissue-accretion  occurs.  The  experi- 
mental indications  are  quite  the  reverse  and  tend  to  show  that  tissue- 
accretion  is  not  a  cause,  but  may  be  a  consequence  of  lowered  exogenous 
metabolism. 

It  is  clear,  however,  that  adults  may  maintain  themselves  in  nitrog- 
enous and  calorific  equilibrium  upon  a  much  lower  protein  intake  than 
is  customary  in  many  countries,  and  the  question  therefore  arises 
whether  a  restriction  of  the  protein  intake,  particularly  in  the  English- 
speaking  countries,  may  not  be  nationally  and  economically  desirable. 
We  should  be  cautious  in  deciding  this  question  upon  an  insufficiency 
of  evidence.  A  multitude  of  factors  enter  into  the  question  besides 
the  merely  financial  factor.  In  the  first  place  it  may  be  stated  that 
no  harmful  effect  of  a  high  protein  diet  in  normal  persons  has  ever  been 
demonstrated.  No  particular  disease  is  noticeably  more  common 
among  people  accustomed  to  a  high  protein  intake  than  among  those 
accustomed  to  a  low  protein  intake.  On  the  contrary  diseases  traceable 
to  lowered  resistance  of  the  peripheral  tissues,  such  as  Trachoma,  are 
decidedly  more  abundant  among  people  whose  diet  is  deficient  in 
protein,  although  it  must  be  admitted  that  the  dietary  of  these  peoples 
is  probably  deficient  in  other  respects  beside  that  of  protein-content. 
A  high  protein  intake  does  not  throw  a  "load  upon  the  kidneys"  which 
is  deleterious  in  normal  persons,  and  in  any  case  the  "load"  is  very 
easily  lightened  by  a  copious  intake  of  water. 

On  the  other  hand,  taking  Australia  as  an  extreme  instance  of  a 
community  which  is  accustomed  to  a  high  protein  intake,  we  find  from 
the  pre-war  statistics  of  the  Commonwealth  Government  that  the 
Death-rate  was  extraordinarily  low,  nearly  one-half  that  which  prevailed 
in  Italy  and  Austria,  lower  in  fact  than  in  any  other  country  excepting 
New  Zealand,  which  is  also  a  community  of  high  protein  consumption. 
The  Cancer  death-rate  was  intermediate  between  that  of  Italy  and  that 
of  France,  two  communities  each  consuming  far  less  meat  per  capita 
than  the  Australian.1  The  birth-weight  of  Australian  infants  of  British 
parentage  exceeds  that  of  British  infants  born  in  England  by  over  ten 
ounces.2  No  trace  of  deleterious  influence  of  the  high  proportion  of 
meat  in  the  dietary  is  thus  perceptible.  On  the  other  hand  the  diver- 
sity of  climatic  and  social  and  economic  conditions  forbids  us  from 
drawing  the  opposite  conclusion  that  the  high  protein  intake  is  posi- 
tively beneficial. 

It  may  be  pointed  out,  however,  that  an  unusually  low,  and  also  an 
exceedingly  high  rate  of  Exogenous  Metabolism  are  alike  deleterious  to 

1  Official  Year-book  of  the  Commonwealth  of  Australia,  1914. 

2  T.  Brailsford  Robertson:     University  of  California  Publications,  Physiology,  1915, 
4,  p.  207.     Amer.  Jour,  of  Physiol.,  1915,  37,  p.  1. 


582  THE  ANIMAL  BODY  AS  A   MACHINE 

the  general  welfare  and  efficiency.  Physicians  seek  in  some  instances 
to  correct  the  former  condition  by  the  administration  of  thyroid 
extract  or  of  other  preparations  which  are  believed  to  stimulate  metab- 
olism. It  is  quite  possible,  however,  that  the  effects  which  are  desired 
might  also,  in  those  instances  in  which  no  manifest  disease  of  the 
thyroid  is  present,  be  elicited  by  an  adequate  increase  in  the  protein 
intake  of  the  patient.  This  possibility  is  merely  mentioned  in  order 
to  illustrate  the  probable  nature  of  the  effects  and  utility  of  a  protein 
intake  in  excess  of  our  minimum  needs.  We  must  recollect  that  it  is 
not  the  energy-output  which  suffices  merely  to  maintain  life,  to  gain  the 
means  of  living  for  another  day,  which  is  of  genuine  value  in  the  eyes 
of  civilized  mankind.  The  products  of  human  effort  which  we  prize 
are  wholly  the  outcome  of  the  small  surplus  of  energy  which  we  col- 
lectively generate  over  and  above  the  minimum  which  will  support  life 
and  propagate  the  species.  This  small  surplus,  which  is  minute  in 
comparison  with  the  aggregate  expenditure,  is  the  origin  of  all  that  we 
cherish,  and,  even  in  purely  economical  terms,  the  cost  of  its  production 
is  negligible  in  comparison  with  its  value.  In  the  absence  of  any 
evidence  of  deleterious  influence,  a  reasonable  excess  of  protein -intake, 
such  as  that  usual  in  the  United  Kingdom  or  America,  should  not  be 
discouraged  in  advance  of  a  clear  demonstration  that  it  plays  no  part 
in  the  generation  of  efforts  which,  in  the  aggregate,  may  outweigh  the 
costliness  of  the  practice.  It  must  be  admitted,  however,  that  even 
upon  this  basis  it  is  difficult  to  defend  the  extraordinarily  excessive 
meat-consumption  which  has  hitherto  been  customary  in  Australia. 

THE  NORMAL  DIET. 

The  normal  dietary  of  a  variety  of  different  classes  and  occupations 
of  society  in  the  United  States  has  been  investigated  by  Atwater  both 
from  the  standpoint  of  composition  and  that  of  Calorific  Value.  The 
following  table  summarizes  some  of  his  results.  It  must  be  recollected, 
however,  that  the  quantity  of  food  actually  digested,  assimilated  and 
utilized,  was  in  each  instance  a  little  less  than  the  quantity  which  was 
ingested. 

Composition  of  the  diet. 

Calories 
per  day. 

3560 
3605 
3530 
3880 
3705 
3405 
8850 
6905 
5740 
4462 
2910 

3465 


Protein, 

Fat, 

Carbohydrate, 

Occupation. 

grams. 

grams. 

grams. 

Farmers'  families  .      .',..,- 

101 

128 

476 

Mechanics'  families     . 

113 

153 

420 

Professional  families    .      ..  '  V 

110 

136 

442 

Five  college-student  clubs   '  . 

127 

181 

402 

Sixteen  men's  student-clubs  . 

105 

147 

465 

Four  women's  student-clubs 

101 

139 

414 

Stonemason,  hard  work    . 

180 

365 

1150 

Blacksmith,  hard  work     . 

200 

304 

365 

Footballer  .      .      .      .      .  i^. 

181 

292 

557 

Sandow                                   ~ 

244 

151 

502 

Teacher's  families,  Indiana   . 

111 

110 

349 

Official's    families,    Pennsyl- 

vania 

98 

155 

396 

NORMAL  DIET  583 

It  will  be  observed  that  the  habitual  performance  of  hard  physical 
labor  is  correlated  with  a  high  calorific  intake.  The  increase  of  intake 
affects,  as  a  rule,  all  three  classes  of  foodstuffs.  The  increase  of  the 
Protein  intake  is  surprising  in  view  of  the  fact  that  proteins  are  not 
a  normal  source  of  muscular  energy.  This  apparent  contradiction, 
which  has  been  observed  in  all  countries,  has  been  explained  in  several 
diverse  ways.  Advocates  of  a  high  plane  of  protein  nutrition  have 
advanced  the  tendency  to  increased  consumption  of  protein  by  those 
who  perform  hard  physical  labor,  as  evidence  that  the  increased  speed 
of  metabolism  induced  by  protein  facilitates  the  functional  activity  of 
the  tissues,  including  muscular  tissues.  Advocates  of  the  low  plane  of 
protein  nutrition,  on  the  contrary,  have  urged  that  the  high  protein 
intake  of  these  persons  is  essentially  accidental,  arising  simply  from  the 
fact  that  they  ingest  larger  quantities  of  all  foodstuffs  and,  maintaining 
the  normal  admixture  of  the  three  types  of  food  material,  incidentally 
consume  more  protein.  This,  however,  was  certainly  not  true  in  the 
case  of  the  blacksmith  and  the  professional  athlete,  Sandow,  whose 
dietaries  were  investigated  by  Atwater.  A  more  reasonable  suggestion 
than  either  of  the  above  is  probably  that  which  has  been  put  forward  by 
Voit,  that  as  persons  accustomed  to  hard  labor  are  usually  more  muscu- 
lar than  sedentary  individuals,  the  total  protein  intake  required  to 
support  the  greater  quantity  of  Protoplasmic  Tissues,  maintaining  their 
wear  and  tear,  and  at  the  same  time  the  exogenous  metabolism,  is 
greater  than  it  is  in  persons,  even  of  like  weight,  in  whom  a  considerable 
part  of  the  weight  is  made  up  of  adipose  tissues,  for  example.  The 
figures  obtained  by  Atwater  are  certainly  suggestive  from  this  point  of 
view,  for  the  total  mechanical  work  performed  by  Sandow  in  a  brief 
daily  exhibition  and  a  period  of  practice  or  exercise  was  evidently  not 
nearly  equal  to  that  performed  by  a  manual  laborer  in  an  eight-  or  ten- 
hour  day.  But  by  exercises  and  a  mode  of  life  carefully  directed  to 
that  end,  Sandow  had  brought  about  in  himself  an  extraordinary  degree 
of  muscular  development,  far  exceeding  that  of  the  ordinary  laborer, 
in  harmony  with  Voit's  suggestion  we  find  that  his  intake  of  protein 
was  nearly  two  and  a  half  times  the  normal,  while  his  intake  of  fat  was 
normal  and  his  intake  of  carbohydrates  only  slightly  above  the  the 
average. 

It  has  already  been  pointed  out  that  the  vegetable  foodstuffs  are, 
as  a  rule,  distinguished  by  their  relatively  low  content  of  protein. 
This  arises  from  the  fact  that  carbohydrates  assume  a  structural  role 
in  plants  \vhile  in  animals  their  place  as  structural  materials  is  taken  by 
proteins.  It  is  from  this  fact  that  one  of  the  several  objections  to 
the  practice  of  Vegetarianism  arises.  A  purely  vegetable  diet  is,  if 
nitrogenous  equilibrium  is  maintained,  an  exceedingly  voluminous  one. 
The  indigestible  residues  of  cellulose  are  large,  the  feces  very  bulky, 
and  the  fecal  masses  occlude  a  proportion  of  otherwise  digestible  and 
assimilable  materials  which  are  voided  with  them.  The  wastage  in  a 
vegetarian  diet  is  for  this  reason  alone  a  considerable  item.  A  much 


584  THE  ANIMAL  BODY  AS  A   MACHINE 

more  serious  source  of  waste,  however,  is  the  incomplete  utilizability 
of  the  small  proportion  of  protein  which  the  vegetable  diet  does  con- 
tain. We  have  seen  from  the  researches  of  London  (Chapter  XI)  that 
the  intestinal  epithelium  exerts  a  preliminary  selective  action  upon  the 
amino-acids  which  are  submitted  to  it  for  absorption,  rejecting  a 
proportion  of  those  which  are  present  in  unwonted  excess.  Now 
the  proportions  of  the  various  Amino-acids  in  the  proteins  of  vegetable 
origin  differ  very  decidedly  from  those  which  obtain  in  proteins  of 
animal  origin  and  therefore,  on  a  purely  vegetable  diet,  the  arnino-acids 
presented  for  absorption  are  in  abnormal  proportion  to  one  another. 
A  portion  of  the  amino-acids  derived  from' vegetable  proteins  by  diges- 
tion are  therefore  rejected  and  voided  in  the  feces.  The  following 
table  shows  the  percentage  of  the  nitrogen  in  various  types  of  food- 
stuffs which  is  actually  assimilated : 

Percentage  of  nitrogen 
Type  of  food.  actually  assimilated. 

Flesh 98 

Fish .      .      .      .      .      .    -  <"  .      .      .      .  97 

Eggs  .  ' ,  "  .      .      .     -.  95 

Milk .      .....      .      ,      .      94  to  95 

Peas,  Beans •  .      .      .      .     ,.      .      .      .  85 

Corn 83 

Wheat-flour 81 

Rice ,......, •'  •  ;  80 

Potatoes .,.,...     .  78 

The  following  shows  the  relative  proportion  of  wastage  on  a  purely 
vegetable  diet,  an  average  mixed  diet  and  a  high  meat-diet  (Atwater 
and  Langworth) : 

Nitrogen  in  grams  per  day  Percentage 

Type  of  diet.  , — > ^ ^  of  nitrogen 

In  food.         In  urine.       In  feces.  wasted. 

Vegetable  diet    ......      13.8  13.9  3.9  28 

Mixed,  average  meat    ....      19.4  15.6  2.4  13 

Mixed,  large  amount  of  meat        .33.1  24.5  2.9  9 

Even  the  amino-acids  which  fail  to  undergo  assimilation,  however, 
do  not  represent  all  the  wastage  which  occurs  on  a  purely  vegetable 
diet,  for  the  process  of  selection  and  rejection  which  initiates  in  the 
intestine  continues  in  the  tissues,  and  the  rejected  excess  of  unutilizable 
radicals  simply  enters  the  exogenous  metabolism  and,  while  it  is  avail- 
able for  the  production  of  heat,  is  useless  for  the  maintenance  of  the 
integrity  and  repair  or  synthesis  of  tissues.  This  fact  is  very  well 
illustrated  by  the  experiments  of  K.  Thomas,  who,  subsisting  upon  a 
diet  of  starch  and  sugar,  estimated  the  minimal  daily  loss  of  tissue- 
protein  and  then  added  to  his  diet  food  materials  of  various  types  in 
order  to  determine  the  relative  power  of  the  proteins  which  they  con- 
tained to  save  the  body  from  loss  of  tissue-protein,  or,  as  he  terms 
it,  the  Biological  Values  of  the  various  proteins.  The  following  were 
some  of  his  results: 


NORMAL  DIET  585 

BIOLOGICAL  VALUES  OF  VARIOUS  PROTEINS,  ESTIMATED  IN 
TERMS  OF  THE  PERCENTAGE  OF  BODY-PROTEIN  WHICH 
THEIR  INGESTION  WILL  SPARE  FROM  Loss. 


Cow:s  milk 

100 

Casein 

70 

Fish   
Rice  
Cauliflower  . 
Crab-meat    . 
Potatoes        .      . 

...         95 

,      .      .      .         88 
.      .      .      .         84 
.      .      .      .         79 
.      .      .      .         79 

Nutrose      .    .-.     .      . 
Spinach      .      .'.... 
Peas      .      .      .     :  .    .      . 
Wheat-flour     .... 
Cornmeal   

.      69 
.      64 
.      .      56 
.      .      40 
.      .      30 

It  is  evident,  therefore,  that  the  nutritive  value  of  peas,  for  example — 
notwithstanding  their  remarkably  high  protein  content  in  comparison 
with  other  vegetables — is  much  less  than  we  might  infer  from  their 
composition,  and  approximately  double  the  normal  protein  intake 
required  on  a  diet  in  which  peas  and  beans  are  the  only  important  source 
of  protein.  Recollecting  that  peas  and  beans  are  the  only  generally 
available  vegetable  articles  of  diet  in  which  proteins  are  at  all  abundant, 
the  difficulty  of  securing  nitrogenous  and  calorific  equilibrium  upon  an 
exclusively  vegetable  diet  must  be  apparent.  The  herbivorous  animals 
can  accomplish  it  by  eating  an  enormous  bulk  of  food,  for  which  their 
intestines  are  specially  adapted  by  their  length  and  capacity.  A  pro- 
portionately bulky  diet  would  insure  grave  digestive  disorders  in  the 
average  human  being  to  whom  it  was  habitual. 

Even  more  serious  difficulties  than  this,  however,  confront  the  would- 
be  vegetarian.  We  have  seen  (Chapter  XX)  that  certain  constituents 
of  the  diet  which  are  associated  solely  with  Animal  Fats  are  absolutely 
essential  both  for  maintenance  and  for  growth.  These  are  lacking  in  a 
diet  composed  of  customary  articles  which  are  solely  of  vegetable 
origin.  The  fat-soluble  essentials  for  growth  and  maintenance  do  not 
occur  in  the  fatty  tissues  of  plants,  in  seeds  and  fruits,  but  in  the  forage- 
parts.  They  are  acquired  from  these  by  the  herbivorous  animals  and 
stored  by  them  in  their  body-fat.  To  obtain  a  sufficiency  of  these 
substances  from  vegetables  in  our  diet,  we  would  be  compelled  to  con- 
sume an  excessive  quantity  of  vegetable  material  of  very  low  nutritive 
value,  containing  a  very  large  proportion  of  indigestible  residue.  It 
may  therefore  be  stated,  and  experience  seems  to  have  fully  justified 
this  deduction,  that  continued  maintenance  of  weight  and  health,  and, 
above  all,  growth,  are  impossible  of  attainment  by  human  beings  who 
confine  themselves  strictly  to  a  vegetable  diet. 

Happily  there  are  few  people  who  are  so  fanatical  in  their  vegetarian- 
ism as  to  attempt  to  subsist  solely  upon  vegetables,  fruits  and  cereals, 
and  the  so-called  vegetarian  usually  partakes  fairly  freely  of  Milk  and 
Eggs.  On  a  mixed  diet  which  contains  a  good  proportion  of  these 
articles  there  is  no  difficulty  in  securing  a  thoroughly  satisfactory 
nitrogenous  and  calorific  equilibrium,  and  experience  has  demon- 
strated that  a  dietary  of  this  character  may  maintain  a  high  standard 
of  bodily  health  and  vigor.  It  is  not  improbable  that  occasional  indi- 


586  THE  ANIMAL  BODY  AS  A   MACHINE 

viduals  would  positively  benefit  by  adopting  a  dietary  of  this  type. 
Others,  again,  might  not  improbably  find  that  while  it  fully  sufficed 
for  the  maintenance  of  weight  and  health  and  the  satisfaction  of  the 
appetite,  yet  better  digestion  and  improved  well-being  would  be 
attained  on  a  dietary  containing  some  proportion  of  meat.  To  the 
majority,  appetite,  taste  and  habit  apart,  it  would  probably  be  indif- 
ferent which  alternative  was  adopted.  Without  positively  encouraging 
such  dietetic  experiments,  especially  where  children  are  concerned,  the 
physician  will  probably,  unless  there  are  certain  indications  to  the  con- 
trary, do  well  to  allow  a  vegetarian  of  this  type  to  indulge  his  whim.  The 
absolute  vegetarian,  however,  who  declines  even  to  partake  of  milk  or 
eggs,  must  be  solemnly  warned  of  the  danger  he  is  incurring  and  the 
almost  inevitably  unhappy  outcome  of  his  fanaticism,  while  his  children 
should  be  shielded,  if  possible,  from  the  outrage  of  the  perpetration  of 
his  delusion,  and  irreparable  detriment  of  their  bodily  welfare. 

On  the  other  hand  an  exclusive  flesh-diet,  which  has  been  advocated 
no  less  warmly  than  vegetarianism  in  certain  ill-informed  quarters, 
is  only  a  shade  less  undesirable  than  an  exclusively  vegetable  diet.  The 
wastage  again  becomes  very  large  on  account  of  the  stimulation  of 
metabolism  resulting  from  the  high  plane  of  protein  intake,  and  an 
abnormally  large  consumption  of  food  becomes  necessary  to  maintain 
nitrogenous  and  calorific  equilibrium.  The  insufficiency  of  the  carbo- 
hydrate intake  provides  little  of  the  proper  nutriment  for  the  muscular 
tissues,  the  power  of  continued  exertion  is  impaired,  and  the  tendency 
to  certain  types  of  auto-intoxication  is  probably  enhanced.  The  diet 
is  so  completely  digestible  that  the  fecal  bulk  is  too  small  to  maintain 
the  proper  tension  and  tonus  of  the  lower  intestine,  and  the  resultant 
stasis  favors  Intestinal  Putrefaction.  The  abundant  variety  of  mineral 
constituents  contained  in  the  vegetable  items  of  the  customary  dietary 
is  replaced  by  the  relatively  limited  variety  and  quantity  of  mineral 
constituents  in  flesh.  The  high  protein  intake  implies  a  high  sulphur 
intake,  and  therefore  the  formation  of  large  quantities  of  sulphuric 
acid,  which  reduce  the  alkali-reserve  and  impose  a  tendency  toward 
Acidosis. 

On  the  whole,  it  must  be  evident  from  the  above  discussion  that 
the  only  safe  prescription  for  continued  employment  by  persons  of  all 
ages  is  that  which  the  good  housekeeper  instinctively  recommends, 
namely  an  abundant  and  varied  diet.  The  requirements  of  the  body 
are  so  numerous  and  so  varied  in  their  character  and  in  the  sources 
from  which  they  must  be  derived  that  in  our  present  state  of  knowledge 
a  dragnet  policy  of  sweeping  into  the  body  a  large  variety  of  dietary 
articles,  is  the  only  one  which  will  ultimately  ensure  a  sufficient  intake 
of  every  possible  requisite.  All  precise  dietary  prescriptions,  however 
well  supported  by  selected  individual  experiences,  are  premature  where 
the  majority  of  humanity  are  concerned,  and  a  diet  of  half -raw  meat, 
recommended  on  the  ground  that,  being  muscle,  it  must  contribute  to 
our  strength,  should  be  viewed  with  no  less  suspicion  than  a  diet  of 


CALORIFIC  REQUIREMENT  AND  THE  SURFACE-LAW      587 

nuts,  advocated  because  some  of  our  arboreal  ancestors  were  perforce 
accustomed  to  partake  of  these  indigestible  delicacies  rather  freely. 

The  physician,  of  course,  will  find  it  imperative  from  time  to  time 
to  impose  quite  severe  restrictions  upon  the  dietary  of  certain  types  of 
patient,  of  diabetics,  for  example,  or  of  persons  afflicted  with  nephritis, 
or  with  certain  types  of  indigestion,  and  often  he  will  achieve  very  great 
success  by  this  simple  means.  His  very  success,  however,  constitutes 
in  certain  cases  a  positive  danger  to  other  people,  through  the  possible 
conversion  of  his  patient  into  a  dietary  propagandist  seeking  to  pro- 
mulgate a  "system"  arising  out  of  the  measures  which  were  found 
effective  in  bringing  about  the  recovery  of  his  own  health.  A  brief 
but  clear  and  simple  statement  by  the  physician  of  the  precise  object 
of  the  dietary  imposed,  and  its  limited  applicability,  might,  not  infre- 
quently, suffice  to  stifle  a  dietetic  fad  at  its  birth. 

THE  CALORIFIC  REQUIREMENT  AND  THE  "  SURF  ACE-LAW." 

The  average  Starvation-metabolism  of  a  vigorous  man  engaged  in 
light  work  and  weighing  70  kilos  is  about  2240  calories  or  32  calories 
per  kilo.  To  maintain  calorific  equilibrium  this  heat- value  must  be 
contained  in  the  food,  and  a  certain  excess  to  compensate  for  the 
stimulation  of  metabolism  or  Specific  Dynamic  Action  of  foodstuffs. 
On  a  normal  mixed  diet  this  amounts  to  from  11.1  to  14.4  per  cent, 
of  the  starvation-minimum  (Rubner).  This  would  indicate  calorific 
equilibrium  on  an  intake  of  from  2488  to  2562  or,  in  round  numbers, 
2500  calories  or  36  calories  per  kilo  of  body-weight. 

The  total  metabolism  varies  very  greatly  in  different  species  of 
animals,  the  metabolism  per  kilo  being  much  higher  in  small  animals 
than  in  large.  This  may  be  inferred  from  the  relative  consumption 
of  Oxygen  per  hour  and  kilo  body-weight  by  different  species.  The 
following  results  are  cited  after  Rubner: 

Grams  of  oxygen 

Weight,  consumed  per  kilo 

Species.  kilos.  per  hour. 

Calf 115.  0.481 

Sheep 66.  0.490 

Turkey ,     .      .         6.2  0.702 

Dog 5.6  0.902 

Goose 4.6  0.677 

Rabbit 3.43  0.735 

Hen 1.51  0.846 

Duck 1.22  1.382 

Finch  0.025  13.000 

Sparrow 0.022  9.595 

The  greater  metabolism  of  the  smaller  animals  arises,  according  to 
Rubner  and  Richet,  from  the  greater  area  of  external  surface  in  pro- 
portion to  their  volume  which  they  present.  If  the  linear  dimensions 
of  a  solid  are  increased  in  the  proportion  of  1  : 2  the  surface  is  increased 
in  the  proportion  of  1:4,  but  the  volume  in  the  proportion  of  1:8, 
so  that  the  ratio  of  surface  to  volume  falls  to  one-half.  The  surface  of 


588  THE  ANIMAL  BODY  AS  A   MACHINE 

a  regular  solid  varies  as  the  two-thirds  exponent  of  the  volume  or  as 
W!,  if  we  measure  volume  in  terms  of  weight.  Now  Rubner  has 
observed  that  the  metabolism  per  unit  of  Body-surface  is  much  more 
uniform  in  different  species  than  the  metabolism  per  unit  of  body- 
weight.  E.  Voit  has  determined  the  heat-production  in  resting  animals 
of  various  sizes  per  kilo  and  also  per  square-meter  of  surface  with  the 
following  results : 

Calories  produced. 

Per  sq.  M. 

Species.                                                     Weight  in  kilos.  Per  kilo.  surface. 

Horse 441.  11.3                 948 

Pig 128.  19.1  1078 

Man 64.3  32.1  1042 

Dog 15.2  51.5  1039 

Rabbit 2.3  75.1                 776 

Rabbit  (without  ears) 2.3  75.1                 917 

Goose 3.5  66.7                 969 

Fowl 2.0  71.0                 943 

Mouse 0.018  212.0  1188 

The  metabolism  per  kilo  in  these  different  species  displays  the 
greatest  diversity,  ranging  from  11  calories  per  kilo  in  the  horse  to 
212  calories  per  kilo  in  the  mouse.  The  metabolism  per  square-meter 
of  body-surface  is  very  nearly  the  same  in  all  of  the  different  species 
investigated,  ranging,  with  the  exception  of  the  rabbit,  from  900  to 
1200  calories  per  square-meter.  Metabolism  bears  therefore  a  far 
closer  relationship  to  surface  than  it  does  to  weight  and  the  relation- 
ship extends  to  different  individuals  of  the  same  species  and  explains 
in  part  the  high  metabolism  of  infants. 

This  relationship,  which  was  discovered  by  Rubner  in  1883  and 
emphasized  by  Richet  in  1885,  was  at  first  interpreted  to  mean  that 
the  main  factor  governing  metabolism  was  the  rate  of  Radiation  of 
Heat  from  the  surface  of  the  body.  Doubt  was  thrown  upon  this 
interpretation,  however,  by  the  discovery  that  the  production  of  heat 
by  warm-blooded  animals  of  different  sizes  continues  to  be  propor- 
tional to  the  body-surface  even  when  the  temperature  of  the  sur- 
roundings is  uniform  or  nearly  uniform  with  that  of  the  body,  so  that 
the  heat-loss  through  radiation  is  a  negligible  proportion  of  the  total 
energy-production.  On  referring  to  the  preceding  table  it  will  be 
noted  that  in  a  rabbit  deprived  of  its  ears,  although  the  radiating  sur- 
face is  much  diminished,  yet  the  production  of  heat  remains  unaltered. 

Although  the  metabolism  per  unit  of  surface  varies  very  much  less 
than  the  metabolism  per  unit  of  weight,  yet  the  proportionality  of 
metabolism  to  surface-area  is  not  nearly  so  exact  as  many  observers 
have  in  the  past  decades  considered  it  to  be.  Thus  Benedict,  in  a 
critical  examination  of  the  ratio  of  Basal  Metabolism  to  surface  in  eighty- 
nine  men,  sixty-eight  women  and  a  large  number  of  infants  found  very 
marked  deviations  from  the  rate  of  strict  proportionality.  As  Benedict 
has  stated :  "  It  is  obvious  that  any  basis  of  comparison  which  involves 
variations  of  40  per  cent,  with  men,  of  43  per  cent,  with  women,  and 


CALORIFIC  REQUIREMENT  AND  THE  SURFACE-LAW      589 


80  per  cent,  with  normal  infants,  cannot  be  considered  as  a  physiological 
law."  Benedict  draws  attention  to  the  great  importance  of  specific 
Stimulators  of  Metabolism,  which  may  be  contained  in  the  diet  or  in 
the  products  of  the  activity  of  certain  tissues.  Thus  after  prolonged 
severe  Muscular  Exertion  the  metabolism  is  stimulated  for  a  long  period 
following  the  cessation  of  exercise  and  the  consumption  of  foodstuffs 
for  the  production  of  mechanical  work.  Yet  the  ratio  of  bodily  sur- 
face to  volume  has  undergone  no  change  in  consequence  of  the  exercise, 
nor  has  the  temperature  of  the  body  risen.  Whatever  may  be  the 
mechanism  which  brings  it  about  it  is  clear  that  products  of  muscular 
exercise  (and  the  same  is  true  of  acidosis)  induce  a  stimulated  combus- 
tion of  foodstuffs,  and  therefore,  in  the  absence  of  ingested  food,  an 
increased  destruction  of  tissue. 


50 
40 
30 
20 
10 
0 

/ 

\ 

<^-N 

\ 

_S> 

—  — 

—  -i^— 

^•== 

•       '            - 

—                     • 

— 

••                • 

_ 

•              • 

•                — 

===» 

—  —  _ 

——  i^K= 

YRS.  |0      20       30      40       50      60       70      80 
CAL.  PER  SQUARE  METER  PER  HOUR. 

FIG.  50. — Chart,  prepared  by  Du  Bois,  showing  the  basal  metabolism  as  measured  in 
calories  produced  per  square  meter  of  body-surface  per  hour  from  birth  until  the  age 
of  eighty-five  years  in  human  males.  Between  maturity  and  the  eighty-fifth  year 
there  is  a  gradual  fall  in  the  intensity  of  metabolism  of  13  per  cent.  (After  Lusk.) 

Benedict  infers  that  the  total  metabolism,  or  metabolism  at  rest  with- 
out food,  is  determined  by  two  main  factors;  the  first  the  mass  of 
Protoplasmic  Tissues  (parenchyma)  and  the  second  the  variable  concen- 
tration of  specific  Stimulators  of  Metabolism  in  the  tissues.  It  was, 
in  fact,  assumed  by  Voit  that  the  total  metabolism  is  actually  propor- 
tional to  the  mass  of  cellular  as  distinguished  from  Sclerous  Tissues 
in  the  body  and  this  view  is  supported  by  the  steady  decrease  in 
metabolism  which  is  characteristic  of  the  period  between  maturity  and 
old  age  in  man  (Fig.  50).  The  increase  in  basal  metabolism  per  unit 
of  weight  or  surface  which  occurs  to  a  very  striking  degree  during  the 
first  year  of  post-natal  growth  is,  however,  only  to  be  interpreted  by 
also  taking  into  consideration  the  second  factor  suggested  by  Benedict, 
namely  the  variable  concentration  of  stimulators  of  metabolism  which 
determines  the  Metabolic  Rate  of  the  tissues.  Ihe  rise  in  metabolism 
which  occurs  in  early  growth  and  just  before  puberty,  therefore,  indi- 
cates an  accumulation  of  stimulators  of  metabolism  which  are  not 
improbably  the  Endogenous  Catalyzers  of  growth. 


590  THE  ANIMAL  BODY  AS  A   MACHINE 

We  must  still  admit  that  the  ratio  of  basal  metabolism  to  surface, 
although  variable,  is  much  less  variable  than  the  ratio  of  metabolism 
to  weight,  length,  temperature,  or  any  other  dimension  or  characteristic 
of  the  individual.  The  possibility  has  not  been  sufficiently  considered, 
however,  that  many  details  of  structural  proportion  in  the  body  may 
be  correlated  with  superficial  area  rather  than  with  weight,  and  that 
the  observed  relationship  of  metabolism  to  surface  may  be  thus  only 
an  indirect  one,  representing  a  relationship  of  metabolism  to  a  group  of 
structural  elements  which  vary  as  the  two-thirds  exponent  of  the 
body-weight  or  volume.  Thus  Dreyer  has  shown  that  the  blood- 
volume  and  the  sectional  areas  of  the  aorta  and  the  trachea  of  animals 
of  different  size  are  proportional  to  W*,  that  is,  to  the  surface.  Frieden- 
thal  has  pointed  out  that  the  sum  of  the  non-protoplasmic  materials 
(reserve-materials,  skeletal  constituents  and  fibrous  tissues)  in  the 
animal  body  increases  more  rapidly  with  total  size  than  the  proto- 
plasmic tissues.  This  is,  in  fact,  inevitable,  for  the  need  of  binding  and 
supporting  tissues  increases  in  proportion  to  the  strains  to  which  the 
body  is  subject  and  these  increase  not  only  in  proportion  to  the  mass 
but  to  the  mass  X  linear  dimensions  of  the  body.  A  small  mass  of 
protoplasm  requires  no  binding  tissues  to  support  it,  but  a  large  mass 
of  cells  would  collapse  of  their  own  weight  without  binding,  cementing 
and  supporting  tissues,  and  the  greater  the  distance  of  any  mass  of 
protoplasm  from  the  center  of  gravity  of  the  whole,  the  greater  in  that 
proportion  will  be  its  tendency  to  break  away.  Friedenthal  concludes, 
in  fact,  that  the  protoplasmic  or  Parenchymatous  Tissues  only  increase 
in  proportion  to  the  two-thirds  exponent  of  the  total  weight,  i.e.,  in 
proportion  to  the  surface.  Since  these  are  the  tissues  of  highest 
metabolic  rate,  their  mass,  together  with  the  proportion  of  Endogenous 
Catalyzers  which  they  contain,  might  be  expected  to  play  a  leading  part 
in  determining  the  rate  of  basal  metabolism. 

THE  NUTRITION  OF  CHILDREN. 

During  the  early  period  of  post-natal  -development  the  sole  normal 
source  of  food  among  the  mammalia  is  Milk.  The  milk  of  different 
species  of  animals,  however,  is  very  far  from  being  of  constant  composi- 
tion, and  we  may  infer  that  the  optimal  admixture  of  foodstuffs  for 
sucklings  varies  greatly  with  the  species.  The  following  table  repre- 
sents the  composition  of  milk  of  several  species,  determined  by  Abder- 
halden. 

One  hundred  parts  by  weight  of  milk  contain : 

Species.  Casein.           Albumin.     Total  protein.           Fat.  Sugar. 

Dog 4.8  2.6  7.4  11.6  3.2 

Pig 3.8  1.5  5.2  9.5  3.3 

Sheep 4.1  0.8  4.9  9.3  5.1 

Goat 2.9  0.8  3.7  4.3  3.6 

Guinea-pig      ...4.8  0.6  5.4  7.0  2.0 

Cow 2.9  0.5  3.4  3.7  5.0 

Horse 1.3  0.8  2.1  1.1  5.9 

Ass 0.8  1.1  1.9  1.4  6.2 

Human     .      .     .     .  0.8  1.2  2.0  3.7  6.4 


NUTRITION  OF  CHILDREN  591 

Human  milk  contains  more  Albumin  and  much  less  Casein  than  cow's 
milk.  This  may  be  only  one  among  many  reasons,  not  readily  deter- 
minable  by  analysis,  why  Artificially-fed  Infants  rarely  thrive  as  well 
as  breast-fed  infants.  This  fact,  which  has  so  often  been  demonstrated 
and  in  such  a  diversity  of  ways,  may  be  illustrated  by  the  following 
tabular  comparison  of  the  growth  of  South  Australian  male  infants 
which  were  in  every  respect  normal,  but  which  in  the  one  group  were 
fed  for  at  least  the  first  few  weeks  at  the  breast,  while  in  the  other  group 
modified  cow's  milk  was  the  source  of  nutriment: 

Average  weight  in  ounces  of 
South  Australian  male  infants. 


Age  in  months. 
1 

Breast-fed. 
155 

Bottle-fed. 
117 

2   
3 

187 
.                                              206 

141 
169 

4   . 

224 

193 

5   . 

254 

226 

6  . 

270 

242 

7  ... 

287 

267 

9  . 

311 

280 

The  nutritional  requirements  of  children  are  much  greater  in  pro- 
portion to  their  weight  than  those  of  adults.  The  heat-production  of 
infants  at  various  ages  is  thus  summarized  by  Murlin: 


Heat  production  of  infants  recently  fed 
and  sleeping. 


Calories  per  square- 
Calories  per  kilo  meter  of  surface 
Age.                                                                           and  hour.  and  hour. 

Birth 1.87  25 

2  to    4  months 2.38  35 

6  to  12  months 2.45  42 

Underfed  and  atrophic  infants  produced  more,  and  overweight 
infants  less  than  the  heat-output  of  normal  infants.  It  must  be 
remembered,  however,  that  these  figures  are  subject  to  considerable 
modification  by  a  variety  of  factors,  among  which  Exercise,  for  example 
crying,  the  type  and  quantity  of  Clothing  worn  and  the  Temperature 
of  the  surrounding  atmosphere  are  the  most  important. 

The  Heat-production  per  kilo  body- weight  in  an  infant  during  the  first 
year  is  about  80  calories,  while  that  of  an  adult  does  not  exceed  36 
calories  per  kilo.  The  heat-production  of  the  Newborn  Infant  is  much 
less  than  at  later  months,  in  many  cases  not  exceeding  48  calories  per 
kilo.  The  heat-production  per  square-meter  of  surface  also  rises  during 
the  first  year.  The  allowance  of  100  calories  per  kilo  which  is  adopted 
by  many  physicians  upon  the  basis  of  the  older  estimations  of  Heubner 
is  undoubtedly  excessive  for  the  average  infant.  Even  taking  80 
calories  per  kilo  as  a  basis,  however,  the  food  required  by  an  infant  of 
10  kilos  at  one  year  of  age  is  one-third  of  that  required  by  an  adult 
weighing  seven  times  as  much. 

This  high  food-requirement  arises  from  three  sources:  Firstly  the 
high  average  Metabolic  Rate  and  the  high  proportion  of  Parenchymatous 


592  THE  ANIMAL  BODY  AS  A  MACHINE 

Tissues  in  young  animals,  secondly  the  larger  proportion  of  surface  to 
volume  involving  a  greater  Radiation  of  Heat  than  in  the  adult,  and 
thirdly  the  energy  absorbed  in  the  building  up  and  retention  of  new 
tissue.  In  older  children  we  must  add  to  these  the  incessant  Muscular 
Activity  which  characterizes  a  healthy  child.  Taylor  states  that  a 
resting  boy  of  ten  years  should  have  a  metabolism  of  about  40  calories 
per  kilo  per  day,  but  when  engaged  in  play  the  diet  of  the  child  may 
have  to  be  as  high  as  100  calories  per  kilo  per  day  to  maintain  calorific 
equilibrium.  "The  diet  of  a  child  must,  therefore,  cover  the  basal 
metabolism,  the  natural  increment  of  growth,  and  the  enormous  output 
for  physical  exercise.  It  is  the  inability  to  judge  these  fractions 
correctly  that  is  responsible  for  so  much  underfeeding  of  children. 
There  are  furthermore  the  additional  deprivations  so  often  inflicted 
on  children  by  the  application  of  fad-notions  of  diet.  The  relative 
caloric  input  of  a  normal  child  leading  an  outdoor  life  is  to  be  compared 
to  that  of  a  man  at  heaviest  physical  work.  Protein  in  excess  is  not 
needed,  that  is  clear;  but  total  calories  are  needed,  in  the  form  of  sugar 
and  fat."  The  craving  of  healthy  children  for  sugar  is  therefore  the 
expression  of  a  normal  and  healthy  need  arising  from  the  high  con- 
sumption of  glucose  by  the  Muscular  Tissues.  It  should  be  satisfied 
by  a  discreet  allowance  of  sugar  and  an  abundant  allowance  of  poly- 
saccharides. 

THE  ENERGY-EQUIVALENT  OF  GROWTH. 

The  storing-up  of  tissue-substance  which  is  possessed  of  a  definite 
calorific  value,  necessarily  results  in  the  retention  by  the  growing 
animal  of  a  proportion  of  the  energy- value  of  its  food,  and,  furthermore, 
a  considerable  proportion  of  the ,  heat- value  of  the  diet,  varying  with 
age  and  the  rate  of  growth,  is  additionally  consumed  and  dissipated  in 
performing  the  work  of  storage.  This  is  doubtless  attributable  to  the 
fact  that  at  all  stages  of  growth,  as  at  all  stages  of  any  chemical  trans- 
formation, the  forward  and  reverse  reactions  are  proceeding  side  by 
side.  In  growth  the  products  of  the  reverse  reaction  (tissue-degrada- 
tion) participate  in  a  side-reaction  (Exogenous  Metabolism)  and  are  thus 
partially  consumed  and  their  energy- value  dissipated  in  the  form  of 
heat,  mechanical  work,  and  the  energy-values  of  the  excreta.  Hence 
we  find  that  in  a  given  species  of  animal,  the  slower  the  accretion  of 
tissue  the  greater  the  energy  consumed  per  kilo  of  tissue  built  up,  since 
the  reverse  reaction  in  such  a  case  is  proceeding  for  a  longer  time.  The 
following  are  illustrative  results  obtained  by  Aron: 

GROWTH  OF  DOGS. 

Calories  consumed 

Animal  Calories  consumed     Increase  in  weight  per  gram  of 

number.  in  fifty  days.  in  fifty  days.  tissue-increase. 

B 19,950  1570  12.7 

C 13,925  1000  13.9  . 

VIII 9,500  780  16.4 

XII 10,750  838  15.6 


ENERGY-EQUIVALENT  OF  GROWTHS 


593 


From  these  results  it  is  clear  that  an  animal  gaining  1000  grams 
in  fifty  days  needs  fewer  calories  for  this  gain  than  one  gaining  1000. 
grams  in  one  hundred  days,  the  reason  being,  as  indicated  above,  that 
in  the  former  instance  the  animal  needs  to  be  "maintained"  for  only 
one-half-as  long  as  the  latter.  The  following  are  comparable  observa- 
tions made  by  Aron  upon  Filipino  children: 


From 
week. 

21 
26 
31 


From 
week. 

4 

9 


To 


Number 


MARIA  INOCENCIA. 

Increase  in  grams. 


Calories. 


week.        of  days. 


26 

31 
35 


To 
week. 

9 
13 


35 

31 
28 


From 
3500 
3650 
4225 


to  Per  day. 
3600  3 

4225          17 
4811          21 


MIGUELO  PRIEGA. 

Increase  in  grams. 

Per  day. 
17 
24 


Number 

of  days.  From  to 

35  3550  4175 

28  4175  4850 


Per  day.  Per  kilo. 

350  to  375  100  to  105 

450  115  to  120 
500  125 


Calories. 


Per  day.  Per  kilo. 

350  to  400  100 

450  to  475  105 


Hence,  during  the  entire  period  of  the  investigation,  Maria  Inocencia 
increased  in  weight  at  the  rate  of  14  grams  per  day  and  consumed  an 
average  of  450  calories  per  day  and  115  calories  per  kilo.  Miguelo 
Priega,  on  the  other  hand,  increased  in  weight  at  the  rate  of  21  grams 
per  day  and  consumed  about  the  same  number  of  calories  per  day  and  a 
considerably  smaller  number  per  kilo. 

According  to  Rubner,  the  energy  consumed  per  kilo  in  doubling  the 
Birth-weight  of  animals  is  always  very  nearly  the  same,  excepting  in  the 
case  of  man,  namely  about  4000  calories.  The  following  data  are 
presented  by  Rubner  in  support  of  this  thesis : 


Species. 
Horse  . 
Cow  . 
Sheep  . 
Hog  . 
Dog  . 
Cat  . 
Rabbit 


Energy-consumption  per  kilo 
in  doubling  the 
birth- weight. 

.  .  .  4512 

.  .  .  4243 

.  .  .  3926 

.  .  .  3754 

.  ,  .  4304 

.  .  .  4554 
5066 


Man  .   28864 


The  generalized  form  of  this  relationship  would  be: 

E 

=     a  log  x   +  b 

where  "E"  is  the  energy-consumption,  "x"  the  weight  of  the  animal 
and  "a"  and  "b"  are  constants  which  are  the  same  for  all  species 
(excepting  man).  Doubling  of  the  weight  would  obviously  always 

add  an  equal  amount  to  the  quotient   -,  that  is,  to  the  total  energy- 

X 

consumption  per  kilo.  This  leads  to  the  differential  or  velocity- 
equation  : 

dE  E 


38 


594  THE  ANIMAL  BODY  AS  A  MACHINE 

which  means  that  the  consumption  of  energy  per  unit  of  tissue-accre- 
tion increases  in  proportion  to  the  energy  which  has  already  been 
consumed  in  reaching  the  weight  to  which  this  unit  of  tissue  is  added. 
When  this  rate  of  energy-consumption  becomes  equal  to  or  less  than  the 
Basal  or  Maintenance-metabolism  it  is  obvious  that  growth  must  cease. 
The  more  rapidly  growth  occurs,  however,  the  less  energy  derived  from 
exogenous  metabolism  is  expended  during  the  time  consumed  in  build- 
ing up  a  unit  of  tissue  at  a  slower  rate.  This  obviously  corresponds 
very  well  with  the  facts  ascertained  by  Aron. 

REFERENCES. 

GENERAL : 

Taylor:     Digestion  and  Metabolism,  Philadelphia,  1912. 

Krogh:     The  Respiratory  Exchange  in  Animals  and  Man,  London,  1916. 

Lusk:     The  Science  of  Nutrition,  Philadelphia,  1919. 
THE  LAW  OF  CONSERVATION  OF  ENERGY: 

Atwater  and  Benedict:  Metabolism  of  Matter  and  Energy  in  the  Human  Body, 
U.  S.  Dept.  Agric.  Bull.,  136,  1903. 

Benedict  and  Milner:     Ibid.,  Bull.,  175,  1907. 

Benedict  and  Carpenter:     Carnegie  Institute  of  Washington  Pub.  123,  1910. 

Zeller:     Arch.  f.  Anat.  und  Physiol.,  Physiol.  Abt.,  1914,  p.  213. 

Murlin  and  Lusk:     Jour.  Biol.  Chem.,  1915,  22,  p.  15. 
THE  PROTEIN- REQUIREMENT: 

Chittenden:     Physiological  Economy  in  Nutrition,  New  York,  1907. 

Albertoni  and  Rossi:     Arch.  f.  exp.  Path.  u.  Pharm.,  1908  Supplement,  p.  29. 

Hindhede:     Skand.  Ai;ch.  f.  Physiol.,  1912:  27,  p.  87;  1913,  28,  p.  165;  1913,  30,  p.  97. 

Rubner:     Ueber  mod  erne  Ernahrungs  reformen,  Berlin,  1914. 
THE  NORMAL  DIET: 

Atwater:  Storr's  Agric.  Exp.  Station  Ann.  Kept.  No.  9,  1896.  Memoirs  of  the 
Nat.  Acad.  of  Sciences,  U.  S.  A.,  1902,  8,  p.  231.  Ergeb.  d.  Physiol.,  1904,  3,  p. 
497. 

Mombert:     Das  Nahrungswesen,  Jena,  1904. 

Rubner:     Article  in  von  Leyden's  Handbuch  der  Ernahrung,   1903,  vol.  1. 
ENERGY-REQUIREMENTS  AND  ENERGY-OUTPUT: 

Rubner:     Die  Gesetze  des  Energieverbrauchs  bei  der  Ernahrung,  Leipzig,   1902. 

Voit,  E.:     Zeit.  f.  Biol.,  1901,  41,  p.  113. 

Friedenthal:     Centr.  f.  Physiol.,  1910,  24,  p.  321. 

Dreyer,  Ray  and  Walker:     Proc.  Roy.  Soc.  B.  1912,  86,  p.  56. 

Lusk:     Jour.  Biol,  Chem.,  1912-13,  13,  p.  155. 

Fry:     Quar.  Jour.  Exp.  Physiol.,  1913-14,  7,  p.  185. 

Benedict:     Jour.  Biol.  Chem.,  1915,  20,  p.  263. 

Dubois:     Arch,  of  Internal  Medicine,  1916,  17,  p.  887. 
STARVATION: 

Benedict:  The  Influence  of  Inanition  on  Metabolism,  Carnegie  Inst.  Pubs.,  Wash- 
ington, 1907,  No.  77.  A  Study  cf  Prolonged  Fasting,  Ibid.,  1915,  No.  203. 

Cathcart:     Biochem.  Zeit.,   1907,  6,  p;   109. 
METABOLISM  OF  INFANTS: 

Abderhalden:     Zeit.  f.  physiol.  Chen).,  1898-99,  26,  p.  487;  1899,  27,  p.  408. 

Heubner:     Jahrb.  f.  Kinderheilkunde,  1905,  61,  p.  430. 

Aron:     Biochem.  Zeit.,  1910,  30,  p.  207.     Philippine  Jour,  of  Sc.,  1911,  6,  p.  1. 
Berl.  klin.  Wochensch.,  1914,  51,  p.  972. 

Murlin:     Proc.  Soc.  Exp.  Biol.  and  Med.,  1914,  12,  p.  15. 

Pritchard:     The  Infant,  Nutrition  and  Management,  London,  1914,  p.  71. 

Benedict  and  Talbot:  The  Gaseous  Metabolism  of  Infants,  Carnegie  Inst.  of  Wash- 
ington, Pub.  201,  1914.  The  Physiology  of  the  Newborn  Infant,  Ibid.,  233,  1915. 
Am.  Jour.  Diseases  of  Children,  1914,  8,  p.  1. 

Murlin  and  Hoobler:     Ibid.,  1915,  9,  p.  81. 
VEGETARIANISM  : 

Atwater  and  Langworthy:     A  Digest  of  Metabolism  Experiments,  Washington,  1898. 

Ostertag:     Handbuch  der  Fleischbeschau,  Stuttgart,  1899. 
Thomas:     Arch.  f.  Anat.  u.  Physiol.,  Physiol.  Abt,,  1909,  p.  219. 

McCollum  and  Davis:     Jour.  Biol.  Chem.,  1913,  15,  p.  167. 

McCollum,  Simmonds  and  Pitz:     Am.  Jour.  Physiol.,  1916,  41,  p   333. 


THE  OUTLOOK. 

The  acquisition  of  knowledge  always  results  in  the  revelation  of 
wider  and  yet  wider  prospects  tempting  inquiry  and  inviting  explor- 
ation. To  the  Pythagoreans  life  and  the  universe  were  fairly  simple, 
a  few  rules  when  once  discovered  would,  they  felt  sure,  reduce  the 
seeming  chaos  to  order.  In  the  laws  of  number  lay  the  simple  clue  to 
the  whole  riddle.  To  Descartes,  two  thousand  years  later  in  the  history 
of  man  and  of  science,  how  much  more  complex  did  the  world  appear. 
Yet  even  he  thought  that  the  phenomena  of  life  could  be  interpreted 
by  geometry  and  hydrostatics  and  that  emotions  arose  through  oscil- 
lations of  the  Pineal  gland,  originating  from  the  varying  pressures  of 
an  impinging  fluid.  But  three-quarters  of  a  century  later,  Newton, 
incomparably  the  greatest  discoverer  of  his  age,  gazed  in  awe  and 
humility  upon  the  limitless  prospect  which  his  labors  had  revealed: 
"  I  do  not  know  what  I  may  appear  to  the  world,  but  to  myself  I  seem 
to  have  been  only  like  a  boy  playing  on  the  seashore,  and  diverting 
myself  in  now  and  then  finding  a  smoother  pebble  or  a  prettier  shell 
than  ordinary,  while  the  great  ocean  of  truth  lay  all  undiscovered  before 
me. ' '  A  new  ocean  of  undiscovered  truth : — that  is  the  revelation  which 
we  glean  from  every  fresh  achievement  of  the  scientific  method,  and 
this  is  essentially  its  most  inspiring  outcome. 

It  is  refreshing,  from  time  to  time,  to  pause  amid  the  fruits  of  our 
collective  labors  and  gaze  upon  the  widened  prospect  which  lies  before 
us,  striving  to  make  out  the  dim  form  of  truths  which  are  emerging, 
half- veiled  in  the  mists  of  the  early  dawn  of  knowledge,  upon  the 
horizon  of  our  inquiries. 

In  the  territory  with  which  we  are  here  most  particularly  concerned; 
that  territory  which  lies  upon  the  borderland  of  life  and  of  atomic 
affinities,  and  seeks  to  illumine  the  one  with  the  beacon-lights  of  the 
other;  the  unexplored  oppresses  us  with  its  vastness  and  entices  us 
with  its  promise,  while  the  known,  the  sure  ground  of  fact,  comprises 
only  the  fringe  of  our  future  heritage  of  knowledge.  In  the  prospect 
which  lies  before  us  certain  objectives  lie  plainly  outlined  and  almost 
within  our  grasp,  others  are  less  clearly  apprehended  and  others,  again, 
loom  gigantic,  unformed,  terrible  in  their  potentialities  for  good  or 
for  evil,  upon  the  ultimate  horizon  of  our  outlook. 

In  the  forefront  of  our  prospect  lie,  patently  enough,  the  vast 
industrial  potentialities  of  our  science,  barely  touched  as  yet,  but 
destined  in  the  near  future  to  be  a  rich  field  of  endeavor,  promising 
inexhaustible  resources  of  wealth  and  power,  the  physical  foundations 
of  intellectual  achievement.  The  accumulated  storehouses  of  fuel, 


596  THE  OUTLOOK 

deposited  in  the  carboniferous  era,  and  now  rendered  available  to  us  in 
the  form  of  coal  and  oil,  have  merely  served,  by  one  of  those  happy  con- 
junctions of  historical  circumstance  which  have  rendered  possible  the 
spiritual  development  of  man,  to  tide  us  over  the  period  of  awakening 
consciousness  and  undeveloped  powers  which  comprised  the  age  of 
steam  and  the  industrial  revolution  of  the  nineteenth  century.  Within 
a  period  which  is  relatively  brief  in  the  age-long  history  of  man  these 
stores  will  be  exhausted  and  we  must,  as  we  assuredly  will,  long  ere 
that  term  arrives,  solve  the  problem  of  manufacturing  illimitable 
supplies  of  fuel.  Ultimately  there  is  only  one  way  in  which  this  can 
be  done,  and  that  is  by  transforming  the  radiant  energy  of  the  sun 
into  the  potential  energy  of  a  falling  weight,  originally  lifted  by  the 
heat  absorbed  in  evaporation,  or  else,  as  in  the  utilization  of  alcohol 
for  motor-fuel,  by  converting  the  radiant  energy  of  the  sun  into  the 
potential  chemical  energy  of  a  carbohydrate  or  a  related  or  derived 
organic  compound.  The  latter  method  lies  almost  within  our  control, 
the  former  not  so  nearly,  and  hence  it  is  to  the  understanding  and 
control  of  the  photochemical  synthesis  of  organic  compounds  that  we 
must  look  in  the  main  for  our  future  sources  of  fuel  and  motive-power. 

The  initial  step  of  photosynthesis  having  been  accomplished,  the 
succeeding  stages  in  the  evolution  of  organic  compounds  in  living 
organisms  are  accomplished  at  low  temperatures  through  the  agency 
of  enzymes.  We  are  gathering  acquaintance  with  the  nature  of  these 
substances  and  of  the  circumstances  and  principles  which  govern  their 
action,  and  through  their  right  understanding  and  employment  we 
will  ultimately  be  enabled  to  accomplish  syntheses  which  at  present 
are  possible  only  in  living  organisms  or,  if  imitable  in  the  laboratory 
may  only  be  achieved  at  the  cost  of  an  expenditure  of  energy  and  raw 
materials  far  exceeding  the  value  of  the  product. 

The  further  investigation  of  the  oxidative  processes  which  occur  in 
living  organisms  and  underlie  luminescence,  is  undoubtedly  destined 
to  supply  us  with  that  hitherto  elusive  ideal,  "cold  light/'  and  the 
remarkable  advances  in  our  knowledge  of  this  field  during  the  past  few 
years,  assure  us  that  this  outcome  of  biochemical  investigation  is  not 
now  very  far  from  practical  realization.  The  meteoric  advance  of 
aviation,  from  the  air-flotation  experiments  of  Langley  to  the  recent 
flight  from  London  to  Australia,  has  shown  us  how  rapidly  in  our 
times  practical  realization  may  follow  upon  the  heels  of  theoretical 
possibility. 

The  fuller  understanding  of  the  nature  of  enzymatic  processes  which 
lies  immediately  before  us  will,  ere  long,  lead  to  the  discovery  of  their 
chemical  nature  and  composition.  Advances  have  already  been  made 
in  this  direction.  Euler  has  produced  an  artificial  oxidase,  and  Falk 
an  artificial  lipase.  It  is  not  at  all  improbable  that  the  digestive 
enzymes  are  not  nearly  so  complex  as  the  earlier  investigators  imagined 
and  that  the  synthesizing  enzymes  are  merely  the  digestive  enzymes 
or  modifications  of  them,  acting  under  differing  physical  conditions. 


THE  OUTLOOK  597 

The  synthesis  and  control  of  artificial  enzymes  will  revolutionize  the 
science  and  art  of  organic  synthesis  and  place  in  our  hands  a  multitude 
of  inestimably  valuable  products  which  have  hitherto  been  regarded 
as  costly  rarities,  the  curiosities  of  a  chemical  museum.  At  the  same 
time,  of  course,  the  production  of  many  substances  which  are  already 
manufactured,  or  derived  from  the  cultivation  of  plants  or  animals, 
will  be  very  greatly  cheapened.  But,  above  all,  the  artificial  produc- 
tion and  the  control  of  enzymes  holds  out  the  hope  of  accomplishing 
the  synthesis  of  foodstuffs  under  conditions  independent  of  climatic 
variations,  and  in  the  immediate  neighborhood  of  the  great  centers 
of  population,  thus  eliminating  for  the  great  majority  of  humanity 
the  enormous  addition  to  the  cost  of  food-values  which  is  comprised 
in  the  expense  of  transportation.  The  synthesis  of  palatable  carbo- 
hydrates and  fats,  sufficing  for  a  certain  proportion  of  our  dietary, 
when  we  once  acquire  control  of  the  enzymes,  should  not  present  any 
insuperable  difficulties.  The  proteins  are  a  far  more  complex  problem 
because  of  the  diversity  of  units  of  which  they  are  composed  and  the 
necessity  for  the  provision  of  each  one  of  them,  nor  will  the  synthesis 
of  amino-acids  suffice,  for  while  these  satisfy  merely  nutritional 
requirements  they  are  not  palatable  and  their  ingestion  in  requisite 
amounts  introduces  abnormal  conditions  into  the  alimentary  canal 
which  are  not  well  tolerated.  The  synthesis  of  "protein-sparers"  of 
the  type  of  gelatin,  polypeptides  which  may  be  utilized  with  advantage 
to  reduce  our  protein  ration,  would  doubtless  be  the  first  step  in  this 
direction. 

After,  all,  however,  it  may  well  turn  out  that  the  most  practicable 
way  to  synthesize  enzymes  is  to  permit  organisms  to  make  them  for  us. 
Not  the  complex  organisms  of  the  present-day  farm,  but  unicellular 
organisms  which  we  may  cultivate  in  vats.  We  have  utilized  such 
organisms  since  the  earliest  dawn  of  history  to  make  alcohol  and 
acetic  acid  for  us,  and  at  the  present  day  we  utilize  unicellular  organ- 
isms, yeasts  or  bacteria,  in  the  manufacture  of  bread,  of  cheese,  in  the 
preparation  of  hides  for  tanning  and  other  processes  of  manufacture. 
This  type  of  industry,  which  is  as  yet  barely  in  its  infancy,  has  received 
a  powerful  stimulus  through  the  necessities  created  by  the  war,  and 
while  in  the  allied  countries  a  special  organism  was  utilized  to  manu- 
facture acetone  for  the  preparation  of  explosives,  in  Germany  yeast 
was  cultivated  in  media  consisting  of  inorganic  salts  and  glucose,  as 
a  means  of  manufacturing  protein.  This  protein,  and  the  fats,  poly- 
saccharides  (glycogen)  and  vitamines  which  the  yeast-cell  also  con- 
tains, might  well  be  employed  as  a  desirable  and  palatable  article  for 
human  consumption,  but  the  method  in  which  it  was  chiefly  employed 
in  Germany  during  the  war  appears  to  have  been  as  a  concentrated  feed, 
economical  of  production  and  transport,  for  the  nourishment  of  cattle. 
The  gradual  replacement  of  the  crude  and  wasteful,  but  picturesque 
and  health-giving  processes  of  the  farm,  interwoven  with  our  remotest 
origins  and  endeared  to  us  by  innumerable  historial  associations,  by 


508  THE  OUTLOOK 

the  "sordid"  processes  of  the  factory  may  well  seem  to  many  a  far 
from  desirable  outcome.  The  scientific  investigator,  however,  like  the 
follower  of  a  religious  order,  stays  not  to  inquire  whether  this  or  that 
particular  consequence  of  his  faith  be  immediately  good  or  bad  in 
its  transient  outcome.  We  cling  to  the  faith  that  the  comprehension 
of  nature  will  yield  ultimate  fruits  of  unalloyed  good.  The  forward 
march  of  that  comprehension  cannot  be  stayed  for  the  loss  of  this  or 
that  implement  of  our  intellectual  youth  which  must,  albeit  with 
poignant  regret,  be  discarded  by  the  way.  The  ultimate  triumph  of 
spiritual  over  material  interests,  values,  and  motives,  which  is  the  goal 
of  our  understanding,  will  yield  us  pleasures  upon  another  plane,  as 
incomprehensible  to  us,  perhaps,  as  ours  are  to  the  primitive  savage. 
Furthermore  if  the  factory  is  "sordid,"  that  is,  after  all,  not  the  fault 
of  the  knowledge  that  rendered  manufacture  possible,  but  of  the  de- 
crepit ideals  and  stunted  imagination  of  those  who  utilize  our  knowledge. 
The  social  evils  which  menace  civilization  in  our  day  are  the  indirect 
outcome  it  is  true  of  the  advances  of  scientific  knowledge,  but  the 
responsibility  for  them  rests  upon  the  whole  of  humanity;  they  are 
the  visible  expression  of  defective  ideals,  defective  understandings  and 
defective  information;  they  are  not  of  the  essence  of  knowledge,  nor 
does  the  guilt  of  their  production  oppress  the  soul  of  the  pure  seeker 
after  knowledge.  An  ape  knows  not  how  to  use  fire  nor  the  savage  how 
to  use  edged  tools.  Both  may  hurt  themselves  with  these  things,  but 
does  it  follow  then  that  they  are  bad  or  that  knowledge  of  them  should 
be  eschewed? 

Perhaps,  after  all,  the  substitution  of  the  factory  for  the  farm  may 
restore,  rather  than  detract  from  the  value  of  the  country  to  man. 
Regret  it  as  we  may,  and  long  before  the  factory-synthesis  of  food- 
stuffs has  begun  to  be  a  measurable  item  in  our  commerce,  the  attrac- 
tivenesss  of  agriculture  as  a  career  is  diminishing  and  has  already 
fallen  far  below  its  ancient  standard.  The  restoration  of  our  country- 
side to  untamed  nature  may  serve  us  after  all  in  good  stead,  and  set 
free  for  us  the  means  of  enjoying  some  of  the  pleasures  of  primitive 
man  once  more,  of  regaining  some  of  the  youth  of  the  world  with  the 
intellectual  heritage  and  the  securities  of  an  old  and  complex  civil- 
ization. 

Returning,  for  the  moment,  to  more  immediately  realizable  possi- 
bilities, the  utilization  of  the  various  products  and  constituents  of 
living  matter,  apart  from  the  foodstuffs,  is  as  yet  in  its  infancy.  The 
value  of  materials  arises  out  of  their  peculiar  suitability  for  the  purposes 
of  man,  on  the  one  hand,  and  their  rarity  on  the  other,  and  the  desires 
and  purposes  of  man  are  so  multifarious  in  their  variety  that  it  may 
be  said  that  any  material  possessed  of  unique  physical  characteristics 
will  ultimately  be  found  of  peculiar  utility  in  satisfying  some  one  or 
other  of  our  needs.  Now  among  the  products  of  vegetable  and  animal 
life,  there  are  numerous  substances  which  are  distinguished  by  their 
possession  of  unique  physical  characteristics.  The  peculiar  properties 


TJJE  OUTLOOK  599 

of  rubber  and  of  the  gums  and  mucilages,  the  adhesive  quality  of  gela- 
tin, the  glaze  communicated  to  surfaces  by  colloids  in  general  and  starch 
and  dextrins  in  particular,  and  the  hard  surfaces  communicated  by  the 
drying  oils  are  already  utilized  in  a  multitude  of  ways  in  our  manu- 
factures and  our  daily  affairs;  but  the  possibilities  held  out  by  the 
products  of  life  are  far  frcm  being  exhausted  by  these  few  instances. 
Among  the  proteins,  for  example  we  find  elastin,  distinguished  by  its 
possession  of  the  rare  combination  of  elasticity  and  tensile  strength 
without  rigidity,  spongin  exhibiting,  although  in  a  different  way,  a 
similar  combination  of  qualities,  keratin,  distinguished  by  its  hardness, 
insolubility,  translucency  and  ability  to  take  a  polish,  fibroin  distin- 
guished by  its  extraordinary  tensile  strength,  lightness  and  insolubility. 
These  few  examples  suffice  to  show  us  what  a  variety  of  physical  char- 
acteristics the  various  proteins  may  display,  and  since  these  substances 
do  not  differ  profoundly  from  one  another  in  structure  and  compo- 
sition, we  may  infer  that  a  relatively  slight  chemical  change  may  confer 
upon  a  protein  an  entirely  new  series  of  physical  characteristics.  An 
example  of  this  is  afforded  by  the  effect  of  union  with  formaldehyde 
upon  the  physical  characteristics  of  casein. 

The  proteins  are,  at  present,  sparingly  employed  in  the  manufac- 
tures, but  casein  is  used  as  a  substitute  for  celluloid,  and  buttons, 
hair-combs,  billiard-balls,  and  other  objects  formerly  made  of  ivory  or 
celluloid  are  now  made  of  casein  rendered  horny  in  consistency  by 
treatment  with  formaldehyde  or  calcium  hydroxide.  Casein  is  further- 
more utilized  as  a  vehicle  for  pigments  in  paints,  as  a  finishing  and 
water-proofing  material,  and  for  the  manufacture  of  non-inflammable 
moving-picture  films.  The  uses  of  gelatin  are  manifold  and  well- 
known.  The  employment  of  the  relatively  expensive  proteins  of  animal 
origin  in  the  manufacturing  industries,  however,  is  excessively  wasteful 
and  cannot  continue  indefinitely,  or  expand  to  very  great  dimensions. 
We  must  seek  substitutes  for  the  proteins  already  used,  and  new  utilities 
as  well,  among  derivatives  of  the  relatively  inexpensive  vegetable 
proteins.  The  exigencies  of  the  war  have,  in  fact,  already  called  into 
being  a  vegetable  glue,  and  a  vegetable  substitute  for  casein  undoubt- 
edly merely  awaits  the  seeker. 

In  agriculture,  our  recent  acquisitions  of  knowledge  in  the  field  of 
growth  have  already  profoundly  influenced  our  practice  in  the  feeding 
of  stock  for  the  market  and  for  breeding  purposes.  Further  advances 
in^this  direction,  together  with  precise  knowledge  of  the  time-relations 
of  growth  in  the  various  domesticated  animals,  will  ultimately  enable 
us  with  the  utmost  precision  to  define  the  most  economical  practice 
of  feeding  and  the  optimal  duration  of  growth  for  the  production  of 
calorific  and  nitrogenous  values.  In  the  growth  of  perennial  crops, 
also,  an  exact  knowledge  of  the  time-relations  of  the  growth-process 
will  enable  us  to  determine  with  precision  the  optimal  period  of  growth 
which  should  elapse  before  cropping.  Especially  in  forestry  this  knowl- 
edge will  increase  the  economy  of  our  practice. 


600  THE  OUTLOOK 

The  biochemical  relations  between  the  soil  and  its  bacterial  flora 
on  the  one  hand  and  the  crop  on  the  other  is  already  a  flourishing 
field  of  investigation,  and  the  results  of  these  inquiries  have  led  to 
very  important  improvements  in  agricultural  practice.  The  further 
development  of  this  field,  and  especially  the  expansion  of  our  knowledge 
of  the  metabolism  and  symbiotic  relations  of  bacteria,  will  point  the 
way  to  a  multitude  of  new  industrial  and  agricultural  applications. 
The  subject  of  plant-pathology  is  also  intimately  related  to  biochemistry 
and  the  investigation  of  the  biochemical  conditions  underlying  gall- 
formation,  for  example,  will  undoubtedly  shed  a  flood  of  light  upon 
the  essential  nature  of  the  internal  factors  which  govern  the  growth 
of  plants. 

It  is  in  the  practice  of  medicine,  however,  that  the  applications 
of  biochemistry  will  ultimately  come  to  affect  human  welfare  most 
directly  and  profoundly.  At  the  present  moment  the  advances  of 
biochemical  knowledge  and  technique  are  rapidly  furnishing  the 
physician  with  diagnostic  methods  of  precision,  and  indications  for 
treatment  based  upon  exact  knowledge,  where  but  a  few  years  ago 
empiricism  afforded  the  sole  basis  of  treatment.  The  discoveries 
which  lie  before  us,  however,  will  ultimately  transform  the  scope,  and 
revolutionize  the  practice  of  medicine,  and  the  substitution  of  knowl- 
edge for  empiricism,  of  science  for  craftsmanship,  as  yet  barely  begun, 
will  not  cease  until  it  is  complete.  The  life  of  man  may  be  regarded 
from  a  material  point  of  view  as  consisting  on  the  one  hand  of  a  struggle 
to  obtain  nutriment,  clothes,  and  other  essentials  of  existence,  and  on 
the  other  hand  a  struggle  to  withstand  the  deleterious  influences  of 
his  environment  and  the  imperfections  of  his  own  organization.  Our 
environment  opposes  us  with  climatic  fluctuations  and  extremes,  and 
with  pervading  toxic  agents,  and  an  ever-present  host  of  parasitic 
organisms  continually  seeking,  and  barely  failing  in  the  conquest  of 
our  tissues.  On  the  other  hand  we  display  the  imperfection  of  our 
organization  in  disorders  of  function  and  in  the  culminating  disorder 
of  senescent  atrophy. 

Each  of  these  disabilities  we  are  seeking  to  conquer  and  in  their 
conquest  and  control  biochemistry  must  necessarily  play  a  leading 
if  not  an  absolutely  decisive  part.  Our  resistance  to  toxic  agents  of 
environmental  or  endogenous  origin  is  rendered  possible  by  a  peculiar 
mechanism  of  adaptation,  or  "  tolerance,"  which  we  as  yet  understand 
very  imperfectly.  Its  understanding  and  control  will  constitute  one 
of  the  most  important  among  the  forthcoming  advances  of  our  knowl- 
edge, and  must  result,  not  only  in  a  greatly  improved  knowledge  of 
the  fundamental  mechanisms  of  adaptation,  but  in  throwing  a  flood  of 
light  upon  pharmacological  science  and  therapeutic  practice.  The 
advances  of  recent  years  have  demonstrated  to  us  that  our  resistance 
to  the  invasion  of  parasites  is  determined  by  specific  chemical  agents 
which  our  tissues  manufacture — the  various  antibodies.  The  chemical 
nature  of  these  substances  is  as  yet  hardly  understood  at  all,  yet  this 


THE  OUTLOOK  601 

knowledge  is  fundamental  to  our  control  of  zymotic  diseases.  We  find 
that  whereas  to  certain  organisms  we  oppose  an  impenetrable  resistance, 
to  others  our  resistance  is  very  slight.  Our  acquired  resistance,  result- 
ing from  infection  or  artificial  immunization,  varies  between  the  same 
extremes.  The  transient  or  inappreciable  immunity  conferred  by 
immunization  in  many  diseases  lays  us  open  continually  to  their 
inroads  with  resulting  loss  of  life  and  efficiency  which  have  been 
displayed  upon  a  gigantic  scale  in  the  recent  world-wide  scourge  of 
influenza.  The  erection  of  defenses  against  such  plagues,  and  the 
common  infections  of  the  respiratory  or  alimentary  tracts  which  are 
responsible,  in  the  aggregate,  for  so  much  loss  of  effort,  time,  life  and 
efficiency  in  the  world,  wrill  never  be  possible  until  we  understand  the 
underlying  chemical  reasons  why  resistance,  natural  or  acquired,  to 
this  disease  should  be  high  and  permanent  and  to  that,  slight  and 
transient,  and  our  understanding  of  this  will  in  turn  depend  upon  the 
acquirement  of  knowledge  of  the  actual  chemical  nature  of  the  anti- 
bodies and  the  precise  nature  of  the  processes  involved  in  their  inter- 
action with  the  tissues  or  toxins  of  the  invading  parasite.  The  study 
of  these  substances  and  reactions  is  proceeding  apace,  and  a  clear  and 
full  understanding  of  the  mechanisms  of  immunity,  while  perhaps  as 
yet  remote,  will  unquestionably  be  acquired. 

The  conquest  of  zymotic  disease  has  begun,  many  of  the  bitterest 
scourges  of  the  middle  ages  have  disappeared  from  our  lives  never  to 
return,  and  one  by  one  our  parasitic  enemies  are  being  deprived  of 
power  to  mar  or  destroy  our  lives.  Our  disorders  of  function  are 
gradually  becoming  understood,  chlorosis  and  gout  are  disappearing, 
myxedema  may  be  prevented,  such  conditions  as  cretinism  and 
asthma  are  being  traced  to  avoidable  origins,  diabetes  is  coming  under 
control,  and  while  cancer  still  exercises  its  ravages  almost  uncurbed 
that  dark  problem  too  now  presents  some  openings  which  the  forth- 
coming advances  of  our  knowledge  of  the  chemistry  of  growth  will 
undoubtedly  enable  us  to  convert  into  means  of  its  eradication  or 
prevention;  for  the  problems  of  pathological  growth  are  fundamentally 
identical  with  the  problems  of  normal  growth,  and  the  information 
which  sheds  light  upon  the  one  type  of  growth  will  reveal  the  origin 
of  the  other. 

Senescence  alone  remains  untouched,  the  final  triumph  of  nature 
over  the  human  desire  to  live;  but  if  we  can  once  rid  ourselves  of  the 
suggestive  influence  of  age-long  experience  and  view  the  phenomenon 
impersonally,  as  the  culmination  of  a  definite,  understandable  and 
therefore  controllable  process,  we.  will  perceive  that  this  too  must 
ultimately,  fall  under  the  sway  of  human  intellect.  The  indefinite 
prolongation  of  his  own  life  is  the  manifest  destiny  of  man,  and  the 
progress  already  achieved  is  certainly  not  less  than  that  which  had 
been  made  toward  our  conquest  of  the  air  when  Leonardo  da  Vinci 
so  confidently,  and  as  it  then  seemed  so  futilely,  predicted  that  man 
would  ultimately  fly. 


602  THE  OUTLOOK 

The  goal  of  the  biological  sciences  has  been  stated  by  J.  Loeb  to  be 
the  artificial  creation  of  living  matter.  To  this,  too,  we  dare  not 
ascribe  impossibility,  but  its  attainment  seems  at  present  to  be  almost 
certainly  more  distant  than  any  of  the  objectives  we  have  hitherto 
reviewed;  for  our  increasing  knowledge  of  life-phenomena  reveals  to 
us  more  and  more  clearly  that  the  processes  of  life  are  wrapped  up, 
not  merely  with  a  peculiar  admixture  of  unstable  chemical  compounds, 
but  also  with  a  definite  architectural  arrangement  of  these  compounds. 
The  simplest  living  organism  with  which  we  are  acquainted  possesses 
a  definite  structure,  and  even  supposing  our  knowledge  of  the  chemistry 
of  life  to  have  become  so  exhaustive  as  to  permit  the  precise  imitation 
of  the  chemical  constitution  of  living  matter,  its  structural  constitution 
would  still  remain  an  incentive  to  investigation  and  an  obstacle,  but 
not  an  insuperable  one,  to  the  attainment  of  our  ultimate  goal. 

The  slow,  hesitating,  clinging  grasp  of  science,  like  that  of  the  many- 
ten  tacled  denizens  of  the  sea,  cannot  be  loosened  or  evaded.  Through 
many  trials  and  failures,  let  the  superficial  appearance  which  hides  the 
precious  truth  be  as  polished  and  impenetrable-seeming  as  it  may,  a 
flaw  will  be  found,  a  foothold  gained,  and  atom  by  atom,  through 
centuries  if  need  be,  the  very  heart  of  mystery  is  unveiled.  There  is 
not,  nor  ever  can  be  in  our  universe,  anything  which  directly  or  in- 
directly can  be  made  to  assail  the  senses  of  man,  that  his  intellect 
cannot  ultimately  fit  into  the  supreme  architecture  of  the  mind,  and 
there  is  not,  nor  ever  can  be,  one  thing  which  the  intellect  of  man  fully 
comprehends  which  he  cannot  in  some  measure  appropriate  and  employ 
for  the  direction  of  his  own  destinies.  But  in  what  way  will  we  employ 
these  powers?  That,  indeed,  is  a  riddle  to  which  science  can  furnish 
no  solution;  its  answer  lies  hidden  from  our  senses,  in  the  deepest 
recesses  of  the  moral  nature  of  man;  but  the  responsibility  for  the 
choice,  whatever  it  may  be,  rests  not  with  the  scientific  discoverer, 
save  only  in  the  degree  to  which  he  shares  our  common  humanity. 


INDEX  OF  AUTHORS. 


The  numbers  in  heavy  type  refer  to  the  bibliographies. 
A  B 


ABDERHALDEN,  E.,  absorption  of  proteins, 
253;  amino-acids  in  blood,  242;  ammo- 
acid  metabolism,  242;  amino-N  deter- 
mination, 146;  chemical  composition 
of  blood,  337,  364;  chemical  identity 
of  animal  proteins,  331;  composition 
of  milk,  590;  enzyme  specificity,  220, 
226;  infant-metabolism,  594;    iron  in 
foodstuffs,  52 ;  iron  therapy  in  anemia,  | 
356;  localization  of  protein  synthesis,  i 
245;  polypeptides  in  urine,  554;  pyrrole  I 
grouping,  488;  time-relations  in  hydro  1-  j 
ysis,  210 

Abel,  J.,  absorption  of  proteins,  253;  ni- 
trogenous waste  products,  565;  vivi- 
diffusion,  243 

Achalme,  227 

Acree,  227 

Adami,  fluid  crystals,  102;  senescence, 
519 

Adamson,  309 

Aders,  146 

Adler,  519 

v.  Adlung,  561,  566 

Albertoni,  594 

Albu,  52 

Aldrich,  499,  519 

Allen,  degeneration  of  islets  of  Langer- 
hans,  406;  depancreatization,  401;  dia- 
betes, 51,  403,  416 

Alsberg,  92 

Anderson,  105 

Anistchakov,  94,  105 

Armsby,  92 

Armstrong,  75,  92,  222,  226,  227 

Aron,  energy-consumption  in  growth, 
592;  infant-metabolism,  594;  starva- 
tion-metabolism, 502 

Arrhenius,  digestion  and  absorption,  253 ; 
enzyme  action,  226,  425;  hydrolysis  of 
ethyl  acetate,  211;  quantitative  secre- 
tion of  gastric  juice,  251;  transmission 
of  bacterial  spores  by  cosmic  dust,  282, 
435 

Ascoli,  551,  552,  566 

Atkins,  261,  283 

Atwater,  calorific  value  of  diet,  582; 
conservation  of  energy,  594;  normal 
diet,  594 ;  respiration-calorimeter,  571 ; 
wastage  on  different  diets,  584 

Auer,  333 


BACH,  413,  416 

Baeyer,  435 

Bailey,  105 

Bain,  520 

Baker,  283 

Bancroft,  F.  W.,  333,  450 

Bancroft,  W.  D.,  309 

Bang,  105 

Barcroft,  303,  309,  352 

Barger,  186,  200 

Barney,  520 

Bartell,  306 

Batelli,  416 

Baumann,  384 

Bayliss,  214,  226 

Bean,  536 

Beatty,  146 

Bell,  536 

Bendix,  75 

Benedict,  calorimetry,  594 ;  factors  deter- 
mining total  metabolism,  589 ;  ratio  of 
basal  metabolism  to  body-surface,  588; 
specific  stimulators  of  metabolism, 
589;  starvation  -  metabolism,  416, 
594 

Bennett,  520 

Berg,  226 

Bergell,  226 

Bernard,  Claude,  glycosuria,  400;  pan- 
creas, 232;  saline  cathartics,  315;  sugar- 
content  of  liver,  86 

Bernheim,  536 

Berninzone,  227 

Bernstein,  442,  445 

Berthelot,  186 

Bertrand,  186,  412,  413 

Bethe,  523 

Beutner,  309 

Biach,  105 

Biddle,  227 

Biedermann,  312 

Biedl,  389 

Biehler,  146 

Bigland,  309 

Birchard,  150,  153,  172 

Blaauw,  430,  431 

Blackman,  445 

Blair,  Bell,  377,  390 

Blake,  310 

Blasel,  156,  172 

Blish,  384 


604 


INDEX  OF  AUTHORS 


Bloor,  absorption  of  fats,  253;  absorp- 
tion of  fatty  acids,  235;  fat-metabol- 
ism, 416;  fats  in  diabetes,  403,  406 

Bohr,  352 

Bolhner,  146 

Bolin,  413 

Bosworth,  105 

Botazzi,  one-sided  permeability  in  kid- 
neys, 291;  osmotic  pressure  of  sea- 
water  and  tissue-fluids,  262,  283 

Boveri,  462 

Bowditch,  438,  518 

Bradley,  223,  361 

Briggs,  364 

Browder,  470,  505,  520 

Brown,  A.  J.,  209 

Brown,  A.  P.,  356,  359,  364 

Brown,  H.  T..  437 

Brunner,  441 

de  Bruyn,  65 

Bryan/ 535 

Buck,  342,  364 

Buckmaster,  362 

Bugarsky,  172 

v.  Bunge,  20;  hematogen,  45;  hippuric- 
acid  synthesis  in  tissues,  32,  556;  min- 
eral requirement  in  foodstuffs,  36,  52 

Burge,  414,  416 

Burnett,  106,  333;  catalase,  414,  146; 
catalyzers  in  cancer,  520;  influence  of 
temperature  on  muscle-stimulation, 
428;  sea-water  glycosuria,  271 

Burton,  309 

Biitschli,  cell-division,  466;  death,  513; 
structure  of  protoplasm,  308 

Butterfield,  359,  364 

Byk,  435 


Cohen,  419,  445 

Cohnheim,  238 

Conklin,  462,  470 

Cook,  316,  333 

Cooke,  309,  390 
I  Corson-White,  106,  505,  520 
!  Cottrell,  273 

Cramer,  502,  519 
!  Cremer,  227 
I  Croft-Hill,  222,  227 

Curtms,  139 

Gushing,  400,  494,  498,  519 

Cushny,  200 

Cutler,  520 

Czapek,  445 


DAKIN,  arginase,  544,  565;  hydrolysis 
mandelic  acid  esters,  219,  226;  nitro- 
genous waste  products,  566;  oxida- 
tions, 416 

Dale,  186 

Davis,  52,  92,  519,  594 

Delage,  450 

Delprat,  520 

Denis,  94,  241,  253,  390,  566 

Descartes,  595 

Donaldson,  518 

Dore"e,  105 

Douglas,  389 

Drechsel,  545 

Dreyer,  590,  594 

Dubin,  566 

Dubois,  414,  416 

Du  Bois,  512,  594 

Ducceschi,  283 

Dumanski,  309 

Duval,  522,  535 


CAJAL,  522 

Cameron,  153,  536 

Cannon,  chemical  correlation  of  circu- 
lation and  digestion,  390;  pylpric 
sphincter.  372;  suprarenal  function, 
371,  390 

Carlson,  401,  421,  428 

Carpenter,  594 

Carrel,  387,  520 

Cathcart,  absorption  of  proteins,  253; 
anti-enzymes,  227 ;  starvation,  416,  594 

Cattell,  390 

Chalatov,  94 

Chick,  426 

Child,  486,  503,  519 

Chittenden,  579,  580,  594 

Chodat,  413,  416 

Chun,  426 

Clapp,  146 

Clark,  520 

Clark,  E.  B.,  75 

Clark,  G.  W.,  364,  455,  470 

Clark,  W.  M.,  274,  283 

Clausen,  420 


EBBINGHAUS,  529,  532,  535,  533 

Ebstein,  75 

Effront,  226 

Eijkman,  191 

Ellis,  105 

Engelmann,  434,  440 

Epathy,  523 

Erdheim,  387 

Erlanger,  253 

Euler,  226,  346,  413,  416,  596 

Ewald,  421 

Exner,  524,  534 


FALK,  596 
Fano,  283 
Faraday,  568 
Farkas,  283 
Fick,  393 


INDEX  OF  AUTHORS 


G05 


Findlay,  390 

Fine,  416 

Fischer,  E.,  ammo-acids,  132;  amino-N 
determination,  133, 146;  enzyme  speci- 
ficity, 226;  lock-and-key  hypothesis, 
219;  mutarotation,  72;  peptide  for- 
mation, 139-142,  147;  peptide  hydrol- 
ysis, 220;  structure  of  hexoses,  55; 
synthesis  of  glycerose,  53 

Fischer,  M.  H.,  308,  309 

Fitz,  564,  566 

Fitzgerald,  332 

Fleischer,  519 

Folin,  absorption  of  amino-acids,  241; 
absorption  of  proteins,  253;  atophan, 
553;  chemical  correlation  of  circula- 
tion, 390 ;  nitrogenous  waste  products, 
566;  origin  of  creatinine,  545;  sulphur 
excretion,  560;  uric-acid  elimination, 
549;  uric-acid  reagent,  191,  199,  549; 
vitamines,  200 

Fourneau,  140 

Fraenkel,  283 

Fraser,  105 

Friedenthal,  cephalization-factor  and 
life-duration,  517,  519;  energy-require- 
ment and  output,  594;  indicator- 
method,  275,  283 ;  protoplasmic  tissues 
and  body -surface,  590 

Fry,  594 

Fiihner,  369 

Fulk,  253 

Funk,  191,  200,  519 

v.  Fiirth,  cholesterol,  97,  105;  iodothy- 
rin,  384;  melanins,  413;  myosin  and 
myogen,  398 


G 


GABRIELT,  283 

Galeotti,  164,  420 

Gamgee,  351,  364 

Gantor,  427,  445 

Gardner,  94,  105,  492 

Garrod,    alcaptonuria,    559;    cystinuria, 

562-566;  urinary  pigments,  566;  pen- 

tosuria,  75 
Gay,  227,  332,  334 
Gies,  226 
Gilbert,  536 
Givens,  52,  551,  566 
Glikin,  105 
Gobau,  526,  535 
Godlewski,  390,  470 
Goodspeed,  426 
Gortner,  chemical  identity  of  fibrins,  331, 

334;  humin  substances,  384 
Graham,  302 
Gramentzki,  215 
Gray,  390 
Grimaux,  139 
Grund,  75 

Gudernatsch,  486,  519 
Gudzent,  552 


i  Guest,  146 
Guggenheim,  200 
Guldberg,  162,  203 
Gtirber,  328 


HALDANE,  389 

Halliburton,  crystalline  form  of  hemo- 
globin, 360,  364 ;  myosin  clot,  398 

Hamburger,  260,  266,  283 

Hammarsten,  blood-coagulation,  343; 
plasma-clotting,  344;  water  in  tissues, 
255 

Handovsky,  172 

Hanriot,  227 

Hanson,  227,  341,  364 

Harden,  227 

Hardy,  inversion  of  precipitating  ion, 
159,  163;  protein  complexes,  171, 
330;  structure  of  gels,  298-301,  309; 
union  of  acids  and  bases  with  proteins, 
172 ;  viscosity,  309 

Hari,  539,  565 

Harris,  433,  445 

Hart,  519 

Harter,  535 

Hartmann,  520 

Harvey,  artificial  fertilization,  470;  bio- 
luminescence,  414,  416;  surface-layer 
of  cells,  309 

Haskins,  545,  565 

Hasselbalch,  283 

Hedblom,  92 

Hedin,  anti-enzymes,  227;  hematocrit 
method,  266,  283 

Heidenhain,  290,  362 

Hekma,  364 

Henderson,  alkali-reserve,  283 ;  fitness  of 
environment,  282;  neutralizing-power 
of  acids,  277,  283 ;  respiration,  389 

Henriques,  242 

Herter,  556 

Hertwig,  membrane-formation  with  chlo- 
roform, 451 ;  temperature-coefficient  of 
development,  423 

Hertz,  316,  333 

Heubner,  absorption-spectrum  of  hemo- 
globin, 359,  364;  heat-production  in 
infants,  591,  594 

Hewlett,  253 

Hildebrandt,  227 

Hill,  303,  309 

Hindhede,  594 

Hinkins,  227 

Hirschfeld,  156,  172 

Hirschstein,  164 

Hoagland,  273,  283,  416,  556,  566 

Hocker,  306 

Hoeber,  262,  274,  283 

Hofmeister,  dehydration  in  protein  coag- 
ulation, 165;  protein  assimilation,  238; 
swelling  of  protein  jellies,  309 ;  union  of 
protein-groups,  172 


606 


INDEX  OF  AUTHORS 


Hoobler,  594 

v.  Hoogenhuyze,  creatine-content  of  mus- 
cle, 399,  416;  origin  of  creatinine,  545, 
566 

Hooper,  52 

Hope,  550,  565 

Hopkins,  accessory  foodstuffs,  193;  cal- 
cium in  foodstuffs,  52;  conjugated  ex- 
creta, 566 ;  endogenous  catalyzers,  478; 
growth-substrates,  519;  intestinal 
putrefaction,  560;  pure  proteins  in 
growth  and  maintenance,  490;  uric- 
acid  output  on  purine-ffee  diet,  550, 
565 

Hoppe-Seyler,  lecithin-content  of  em- 
bryonic tissue,  463;  volumes  of  plasma 
and  corpuscles  in  blood,  336 

Horsford,  327 

Howell,  blood-coagulation,  346-348; 
fibrin-structure,  348-349;  hemophilia, 
347;  kephalin,  344;  vasomotor  theory 
of  sleep,  530 

Hoyt,  433,  445 

Hiifner,  359 

Hunt,  385 

Hunter,  allantoin,  551,  566;  protein-pro- 
tein compounds,  170,  172;  uricolytic 
index,  551 

Hurwitz,  341,  364 


IMBERT,  441,  445 
Izar,  551,  566 


JACOBS,  75,  183 
James,  535 
Johansson,  539,  565 
Johnson,  519 
Jolles,  566 
Jona,  262,  283 
Jones,  H.  C.,  165 
Jones,  W.,  146,  182,  200 
Joslin,  403,  407,  416 
Jost,  445 


KANITZ,  influence  of  reaction  on  enzymes, 
226;  temperature-coefficient  of  heart- 
beat, 420,  445 

Kastle,  enzymatic  synthesis,  227 ;  oxidiz- 
ing enzymes,  416 

Katz,  309,  361 

Kellner,  395 

Kendall,  384,  387,  390,  489 

King,  519 

Kleiner,  405 

Klugine,  251 

v.  Knafn-Lenz,  309,  459 


Knoop,  66,  416 

Knudsen,  253 

Kober,  433,  445 

Koch,  119 

Kocher,  390 

Koelker,  210 

Koeppe,  cytolytic  agents,  451;  hemato- 
crit,  266;  origin  of  acid  secretions,  327  ; 
osmotic  pressure  of  cell-contents,  283 

Konig,  381 

Koranyi,  528,  535 

v.  Korosy,  309 

Kossel,  acid-combining  capacity  of  sal- 
mine,  153;  amino-acids,  132;  amino-N 
determination,  146;  arginase,  544;  ni- 
trogenous waste  products,  565 

Krause,  547 

Krogh,  422,  423,  547,  565,  594 

Kiilz,  392 

Kunkel,  52 

Kurijama,  92 

Ktister,  364 


LA  FORGE,  92 

Lamson,  253 

Lander,  105 

Landois,  388 

Landsteiner,  38 

Lane-Claypon,  390 

Langley,  596 

Langworthy,  584,  594 

Lapicque,  52 

Laqueur,  368,  389 

Lavoisier,  567 

Leathes,  332 

Leavenworth,  155,  172 

Le  Bel,  54 

Lee,  439 

Lehmann,  102 

Lepine,  522,  535 

Levine,  amino-N  determination,  146; 
amino-sugars,  75,  92;  chondroitinsul- 
phuric  acid,  92;  glucosides,  75;  glu- 
cothionic  acids,  91;  dinucleotid  link- 
age, 182;  nucleic  acids,  200;  pentoses, 
75;  phospholipins,  119;  thymus  nucleic 
acid,  183 

Levites,  172 

Levy,  283 

Lewis,  166 

Lewkowitsch,  119 

Liebermann,  172 

Liebig,  315,  488,  569 

Liesegang,  309 

Lifschutz,  105 

Lillie,  F.  R.,  artificial  fertilization,  470; 
twin  pregnancy  in  cattle,  377,  390 

Lillie,  R.  8.,  antagonistic  salt  action,  333 ; 
artificial  fertilization,  470 ;  membrane- 
formation,  459;  muscular  contraction, 
445;  osmotic  pressure  of  proteins,  303, 
309 


INDEX  OF  AUTHORS 


607 


Linder,  158,  163 

Lipman,  321,  333 

Loeb,  J.,  antagonistic  salt  action,   318, 
322,  333;  artificial  fertilization,  309, 
446,    470;    Bunsen-Roscoe  law,    431;! 
crystal-form  of  hemoglobin,  359,  364;  i 
cytolysis,    459;    cytolytic    power    of ! 
foreign  blood,  453,  470 ;  effect  of  lack 
of    oxygen     on     development,     460; 
general  physiology,  283;  growth,  518; 
heliotropism,  429-30,  445 ;  immortality  ! 
of  unicellular  animals,  514;  influence 
of   reaction    on   life   phenomena   and 
enzymes,  225,  281;  memory,  535;  oxi- 
dation in  sea-urchin  eggs,   459,   461; 
parthenogenetic  frog,  450;  protoplas- 
mic streaming,  467;  ratio  of  sodium 
to  calcium  in  tissues,  313;  rhythmic 
contraction  in  jellyfish,   313;    Ringer  i 
and  Locke's  solution,  268;  saline  cathar-  ! 
tics,  315;  salt  stimulation,  311;  selec-  j 
tive  action  of  tissues,  329;  senescence,  I 
519;  swelling  in  tissues  and  jellies,  307,  j 
309 ;  synthesis  of  nuclear  material,  463, 
470 ;  time-relations  of  voluntary  move-  ! 
ment,  528;  twin  formation,  470 

Loeb,    L.,    coagulation   of   blood,    364;  j 
growth-catalyzers,  519 ;  placenta!  out- 
growth,   378,  390;  toxicity  of   white 
light,  432;  wound -healing,  520 

Loeb,  W.,  437,  445 

Loevenhart,  227 

Loewy,  554 

London,  digestion  of  proteins,  241;  gas- 
tric digestion,  247;  selective  action  of 
intestinal  epithelium,  584 

Lopez-Suarez,  92 

Lubs,  283 

Lucas,  427,  428,  445 

Ludeking,  309 

Luden,  101,  106,  511,  520 

Lundsgaard,  283 

Lunin,  52 

Lusk,  carbonaceous  waste  products,  565 ; 
dextrose-nitrogen  ratio,  403;    energy-  < 
requirement  and  output,  594 ;  lactic-  ! 
acid  output  in  phosphorus  poisoning, 
398;   metabolism,   416,    594;   specific! 
dynamic  action  of  proteins,  578 

Liithje,  402 

Lyman,  553 


M 


McCLENDON,  294,  309,  470 

McCollum,  essential  dietary  constitu- 
ents, 489;  growth-substrates,  519; 
hippuric-acid  excretion,  556,  566;  in- 
organic foodstuffs,  52;  symbiosis,  92; 
vegetarianism,  594 

McCord,  500,  519 

McDougal,  442,  445 


McKendrick,  475,  478,  482 

McLean,  phospholipins,  119,  345,  349 

McQuarrie,  341,  364 

Macallum,  A.  B.,  iron  in  foodstuffs,  52; 
kidney-development  in  protoverte- 
brates,  270;  mineral  constituents  of 
serum  and  sea-water,  269,  283 

Macallum,  A.  B.  (Jr.),  191,  200,  519 

Macallum,  J.  B.,  effect  of  calcium  remov- 
al on  tissues,  333 ;  emulsions  and  sur- 
face-tension, 309;  saline  cathartics,  316 

Macallum,  W.  G.,  387,  390 

Maclean,  119 

Macleod,  absorption  of  carbohydrates, 
253;  chemical  regulation  of  circula- 
tion, 389;  diabetes,  416;  equilibrium 
between  ammonium  carbamate  and 
carbonate,  545;  glucohemia  from 
splanchnic  stimulation,  371;  glucose 
metabolism  in  depancreatization,  405; 
nitrogenous  waste  products,  565 

Madsen,  213 

Magnus-Levy,  409 

Maly,  327 

Manacelne,  536 

Mandel,  91,  92,  398 

Mansfield,  416 

Marchlewski,  355,  364 

Marcuse,  392 

Marie,  494,  497 

Marshall,  253,  279,  390 

Martin,  364,  426,  519,  540 

Matthaei,  429,  445 

Matula,  156,  172 

Maudsley,  524,  535 

Mawson,  395 

Maxwell,  C.,  445 

Maxwell,  S.  S.,  200,  388,  390,  426,  445, 
499 

May,  119,  200 

Mellanby,  416,  545,  566 

Meltzer,  333,  405 

Mendel,  amino-N  content  of  proteins, 
146;  dietary  essentials,  490,  492,  513; 
growth  substrates,  519;  nitrogenous 
waste  products,  566;  parenteral  ad- 
ministration of  proteins,  239;  protein 
absorption,  253 ;  starvation-metabo- 
lism, 416,  502;  symbiosis,  92 

Mendenhall,  390 

Merckx,  333 

v.  Mering,  401 

Mesernitzky,  463 

Metchnikoff,  514,  519 

Meyer,  G.,  75,  253 

Meyer,  Hans,  293 

Meyer,  K.  F.,  341,  364 

Meyer,  Max,  523 

Meyers,  200,  416 

Michaelis,  283,  308 

Miescher,  463,  470 

Milner,  594 

Minkowski,  401,  543 

Minot,  518,  519,  566 

Mitchell,  519 


INDEX  OF  AUTHORS 


Miyake,  536 
Moll,  536 
Mombert,  594 
Moore,  A.  R.,  309,  470 
Moore,  B.,  309,  424 
Moore,  T.  E.,  428 
Morawitz,  344,  346,  364 
Morgan,  446 
Mofgenroth,  245 
Morgulis,  92 
Morner,  146 
Morris,  437 
Morse,  416 
Mosso,  523,  535 
Muirhead,  565 
Munk,  236,  522,  535 
Murlin,  591,  594 


NAGELI,  330 

Nathanson,  309 

Nencki,  ammonia  retention  by  liver,  544; 
chemistry  of  hemoglobin,  364 ;  urea  ex- 
cretion with  Eck  fistula,  542 

Neubauer,  559 

Neuberg,  75 

Newcomer,  364 

Newton,  595 

Nicolaier,  553 

v.  Noorden,  416 

Du  Nouay,  520 

Nuttal,  92,  331,  334 


OCHORWICZ,  536 

Osborne,  amino-N  determinations,  146; 
copper  compounds  of  edestin,  155; 
growth  on  deficient  diets,  492,  513; 
growth-substrates,  519 ;  inorganic  food- 
stuffs, 52;  oligodynamic  action,  330; 
origin  of  acid  secretions,  328,  334 ;  pure 
proteins  in  growth  and  maintenance, 
490;  starvation,  502;  union  of  acids 
and  bases  with  proteins,  172 

Osier,  337 

Osterhout,  action  of  anesthetics  on  pro- 
toplasm, 326;  antagonistic  salt  action, 
318,  320;  electrical  conductivity  of 
tissues,  294,  322 

Ostertag,  579,  594 

Ostwald,  William,  166 

Ostwald,   518,  536 

Oswald,  384 

Ota,  165 

Overton,  isotonic  solutions,  264;  narcosis, 
293,  309;  one-sided  permeability,  289  ! 


PALITZSCH,  281 

Palladin,  445 

Panzer,  104 

Parkin,  437,  445 

Pasteur,  54,  435 

Paton,  388,  389,  390 

Patrick,  536 

Patten,  146 

Pauli,  acid-combining  capacity  of  pro- 
teins, 156,  172;  coagulation  of  pro- 
teins, 164;  electrolyte-free  egg-albumin, 
164;  swelling  protein  jellies,  309 

Pawlow,  ammonia  retention  by  liver, 
544;  digestion,  390;  enterokinase,  375; 
pancreatic  secretion,  374;  reflex  stimu- 
lation of  gastric  glands,  372;  secretion 
acid  gastric  juice,  328;  urea  excretion, 
542 

Pearce,  389,  405 

Pearl,  519 

Pembrey,  389,  539,  565 

Pfliiger,  402,  416 

Philip,  283 

Piccinini,  420 

Pickering,  166,  343 

Picton,  158,  163 

Pieron,  532,  535,  536 

Pitz,  594 

Plimmer,  147,  463,  470 

Poiseuille,  315 

Porter,  518 

Pottevin,  223 

Poynting,  166 

Pregl,  554 

Pribram,  146 

Priestley,  436,  445,  567 

Prince,  522,  536 

Pritchard,  594 

Procter,  305,  309 


Q 


QUINCKE,  308 


RAMSDEN,  308 
Ray,  106,  519,  520,  594 
Rayleigh,  285,  445 
Read,  B.  E.,  200 
Read,  J.  M.,  472,  519 
Reichert,  356,  360,  364 
Reid,  303,  309 
Reiss,  364 
Rhode,  385,  390 
Richet,  587 
Righetti,  364 
Ringer,  311,  411 
Roaf,  309,  353 
Roberts,  518 


INDEX  OF  AUTHORS 


609 


Robertson,  anti-enzymes,  227;  antigenic 
properties  compound  proteins,  332, 
334 ;  calcium  removal  and  stimulation, 
333 ;  catalyzers  in  cancer,  520 ;  chemi- 
cal mechanics  of  cell-division,  470; 
cholesterol  and  carcinoma,  106;  crys- 
stal-form  hemoglobin,  364;  emulsions 
and  surface-tension,  309;  enzymatic 
synthesis,  226,  227;  factors  determin- 
ing gestation-period,  390;  forgetting, 
536;  general  characteristics  of  pro- 
teins, 147;  growth,  519,  520;  healing  of 
wounds,  520;  influence  of  reaction  on 
enzymes,  226;  influence  of  tempera- 
ture on  life-processes,  445 ;  membrane- 
forming  agent  in  blood,  470;  memory, 
535;  muscular  contraction,  445;  neu- 
trality of  tissues,  283 ;  phospholipins  in 
developing  sea-urchin  eggs,  463,  470; 
precipitation  and  coagulation,  172; 
protein  compounds,  172;  rate  of  ex- 
traction of  colloids,  536 ;  refractometric 
method  for  globulin-albumin  ratio, 
340;  respiration,  389;  serum-proteins, 
364;  tethelin,  119;  viscosity,  309 

Rockwood,  239,  253 

Rogoff ,  390 

Rohmann,  164 

Rohonyi,  301,  309 

Romanes,  313 

Rona,  308 

Rose,  105,  416,  551,  566 

Rosenberg,  359,  364 

Rosenheim,  119 

Rossi,  594 

Rowe,  364 

Rowntree,  200,  253,  283 

Rubner,  calorific  output,  513;  calorific 
value  of  proteins,  570;  energy-con- 
sumption in  growth,  593;  energy- 
requirement  and  output,  594;  equical- 
orific  quantities  of  fats  and  carbohy- 
drates, 575;  isodynamic  value  of  food- 
stuffs, 396;  metabolism  per  unit  body- 
surface,  588;  metabolism  of  small 
animals,  587;  normal  diet,  594;  pro- 
tein requirement,  594 ;  senescence,  519 ; 
specific  dynamic  action,  578;  total 
metabolism  of  different  animals,  587 


SABBATINI,  342,  364 

Sackur,  295,  309 

Saiki,  227,  519 

Salkowski,  561 

Sansum,  253,  361,  416 

Saundby,  519 

Saxon,  106,  505,  520 

Schafer,  endocrine  organs,  200,  389,  519; 

microscopic  structure  of  muscle,  442; 

retardation  of  growth,  499 
39 


Scheele,  107  . 

Schimmelbusch,  348 

Schlesinger,  316,  333 

Schmidt,  A.,  343,  345 

Schmidt,  C.  L.  A.,  action  of  tethelin, 
119,  200;  antigenic  properties  of  com- 
pound proteins,  332,  334;  influence 
of  reaction  on  enzymes,  226;  globin- 
deuteralbumose  compound,  170,  172 ; 
globulin-albumin  ratio  in  protein  im- 
munity, 341;  modification  of  Cottrell 
H  electrode,  273,  283 ;  serum-proteins, 
364;  tables  pn,  H+  and  OH~  values, 
283;  taurine  metabolism,  561,  566 

Schmiedeberg,  32,  315,  556 

Schneider,  389 

Schonbein,  412 

Schottelius,  92 

v.  Schroeder,  309,  544 

Schultz,  158,  160,  163,  361 

Schumm,  359,  364 

Schiitzenberger,  138 

Scott,  389,  463,  470 

Seidell,  193,  200 

Sellards,  283 

Shaffer,  394,  416 

Sharp,  283 

Sherman,  47 

Sherwin,  566 

Shorter,- 309 

Sidis,  522,  536 

Simmonds,  594 

Sjoqvist,  212 

Skraup,  146 

Smith,  499,  519,  535 

Snyder,  420 

Sollmann,  200 

Sorenson,  formol  titration,564;  indicator- 
method,  275,  283 

Soret,  350,  433 

Spain,  520 

Starkenstein,  105 

Starling,  digestion  and  metabolism,  389, 
390 ;  origin  of  lymph,  363,  364 ;  osmotic 
pressure  of  protein  solutions,  302 

Stehle,  52 

Steinach,  376,  390 

Stepp,  519 

Stern,  416 

Stewart,  336,  389,  390 

Stockard,  470 

Stockholm,  385,  390 

Stokes,  351 

Stratz,  519 

Sweet,  106,  505,  520 

Swift,  536 

v.  Szily,  283 


TALBOT,  594 
Tashiro,  427 


610 


INDEX  OF  AUTHORS 


Taylor,  amino-N  determinations,  146; 
digestion  and  metabolism,  416,  594; 
enzymatic  synthesis,  227;  fermenta- 
tion, 226;  protamine  synthesis,  224; 
protein  requirement,  580;  purine  me- 
tabolism, 551,  566;  solubility  of  urates, 
552;  station  of  equilibrium  in  enzy- 
matic hydrolysis,  223;  time-relations 
in  hydrolysis,  210 

Thierfelder,  92 

Thomas,  584,  594 

Thompson,  364,  519 

Thomson,  284 

Tottingham,  200 

Toyama,  364 

Tranter,  364 

Turner,  253 

U 

AF  UGGLAS,  170,  172 
Uhlenhuth,  359,  519 
Underbill,  253 
Usher,  436,  445 


VAN  SLYKE,  absorption  of  proteint,  253; 
acidosis,  564,  566;  alkali-reserve  of 
blood,  279,  283;  ammo-acid  equilib- 
rium in  tissues,  561 ;  amino-N  determi- 
nation, 144;  free  amino-groups  in  pro- 
tein molecule,  150,  153;  hydrolysis  of 
proteins,  147;  union  of  groups  in 
proteins,  172 

Van't  Hof,  enzymatic  synthesis,  222 

Vernon,  155 

Verploegh,  399,  416,  545,  566 

Verzar,  368,  389 

Vincent,  389 

da  Vinci,  Leonardo,  601 

Voegtlin,  200,  390 

Volt,  C.,  518 

Vqit,  E.,  growth,  518;  heat-production 
in  resting  animals,  588,  594;  protein 
assimilation,  238;  protein  metabolism 
in  work,  394;  standard  requirement  of 
proteins,  579;  total  metabolism,  589 

de  Vries,  263 

W 

WAAGE,  162,  203 
Wacker,  106,  511,  520 


Wagner,  315 

Waksman,  119 
!  Walbum,  213 
i  Walker,  594 
I  Waller,  438 
!  Walters,  210 
i  Warburg,  460,  461,  565 
I  Wasteneys,  423,  430,  431,  459,  461,  463, 
470 

Watson,  561,  566 
i  Waynick,  333 

Wells,  253 

Weinland,  227        •  •'••- 

Weir  Mitchell,  51 

Wells,  200,  240,  364 

Weltmann,  105 

West,  119 

Whetham,  160,  172 

Whipple,  absorption  of  proteins,  253; 
fibrinogen  content  of  blood  in  phos- 
phorus- and  chloroform-poisoning,  348; 
iron  as  foodstuffs,  52;  proteose  intoxi- 
cation, 560 

Wichmann,  361 

Wiedermann,  309 

Wiessmann,  514 

Wilder,  253,  416 
i  Willcock,  490, 519 

Williams,  192,  193,  200 

Wilson,  309,  390 
i  Windaus,  66 
!  Winterstein,  368,  369 
i  Wislicenus,  393 
|  Wolf,  441 

Woodruff,  519 

Woodyatt,  253,  416 

Wooldridge,  343,  344,  364 
Wright,  344 
Wuertz,  331,  334 
Wulzen,  499,  519 


YOUNG,  227 


ZALESKI,  364 
Zeller,  576,  594 
Ziehen,  522 
Zsigmondy,  361 
Zuntz,  396 


INDEX  OF  SUBJECTS. 


of  fats,  232,  234 

-spectrum  of  blood,  350 

of  water  from  intestine,  252 
Accelerative  factor  in  growth,  480 
Accessory  foodstuffs,  193 

hydroaromatic  derivatives,  95 
Acetic  acid,  108 

in  butyric-acid  oxidation,  411 
in  diabetic  urine,  405 
in  membrane-formation,  448 
Acetonitrile,  385 
Acetyl  choline,  197 

number  of  fats,  110 
/3-Acetyl-propionic  acid,  174 
Achroodextrin,  86 
Acid  albuminate,  130 

number  of  fats,  110 

secretions,  origin  of,  327 
Acidosis,  279,  564 

ammonia,  utilization  in,  545 

in  children,  409 

in  diabetes,  276,  405 

on  fat-diet,  576 

on  flesh- diet,  586 

urinary  ammonia  in,  276 
Acids,   production   of,   in   brain   during 

activity,  526 

Acree's  reaction  for  proteins,  143 
Acromegalic  gigantism,  496 
Acromegaly,  494,  511 

tethelin  in,  1 18 
Acrpse,  54 

Action-current  in  muscle,  rate  of  con- 
duction of,  428 
Addison's  disease,  369 
Adenase,  177 
Adenine,  174,  178 

in  antineuritic  substance,  193 

formula,  etc.,  176 

mononucleotid,     deaminization     in 

tissues,  231 

Adenine-uracil  dinucleotid,  181 
Adenosine,  178 
Adrenaline,  369,  489,  514 

effect  of,  on  blood-pressure,  187 

effects  of  administration  of,  370 

formula,  etc.,  197 

tests  for,  198 
Adrenin,  197 

Aerobic    bacteria,    oxygen-requirement, 
434 


Agalina,  426 

Agar  jelly,  spongy  structure  of,  298 

Agar-agar,  84 

Agmatine,  physiological  action  of,  187 

Alanine,  134,  398 

0-alanine,  189 

Albino  rabbits,  343 

Albumin  in  blood-serum,  339 

in  milk,  591 
Albuminoids,  127 
Albumins,  126,  136 
Albuminuria,  342 
Albumoses,  132 
Alcaptonuria,  558 

Alcohol    in    protein    coagulation,    122, 
169 

respiratory  quotient,  538 
Alcohol-soluble  proteins,  136 
Alcoholase,  412 
Alcohols,    monatomic,    permeability    of 

blood  corpuscles  for,  268 
Aldoses,  57 

distinction  of,  from  ketoses,  62 
Alexin,  454 

fixation,  333 
Algin,  84 

Alimentary  glycosuria,  72,  229,  400 
Alkali     administration     and    uric-acid 
removal,  553 

albuminate,  130 

Alkali-reserve  of  blood,  273,  279 
Alkaline-earth  chlorides,  in  sensitization 

of  eggs  to  serum,  217 
Alkalinity,  critical,  in  tryptic  digestion, 

217 

i  Alkaloidal  reagents,  122 
Alkalosis  in  parathyroidectomy,  387 
Allantoin,  550 
Allen's  paradoxical  law,  408 
Alloxan,  175 
Alloxantin,  199 
Alveolar  air,  279,  367 
Ambard  formula,  564 
Ambergris,  101 
Amblystoma,  thymus  administration  and 

tetany,  389 
|  Ambrine,  101 

!  Ameba,  protoplasmic  streaming  in,  444 
Amines  derived  from  amino-acids,  184 

produced  by  bacterial  action,  185 
Amino-acetic  acid,  formula  of,  134 

synthesis  of,  by  living  tissues, 
490 


612 


INDEX  OF  SUBJECTS 


Amino-acid  equilibrium  in  tissues  and 

circulation,  561 
radicals  in  proteins,  determination 

of,  144 

lacking  in  certain  proteins,  490 
Amino-acids,  121,  131-138 

absorption  and  assimilation  of,  240- 

246 

amines  derived  from,  184-186 
assimilation-limit  of,  244 
in  blood,  337 
formula?  of,  134-135 
heat-output  of,  578 
proportions   of,    in   vegetable   pro- 
teins, 584 

in  protein  digestion,  240 
ultraviolet  spectrum  of,  433 
a>-Amino-acids,  189 
Amino-benzoic     acid,     detoxication    of 

ultraviolet  light  by,  433 
7-Amino-butyric  acid,  189 
Aminp-ethyl  alcohol,  196 
a-Amino-glutaric  acid,  135 
a-Amino-5-guanidine-valerianic  acid,  135 
a-Amino-iso-caprpic  acid,  134 
a-Amino-iso-valerianic  acid,  134 
a-Amino-j8-methyl-j8-ethylpropionic  aci  d, 

134 

o:-Amino-normal-caproic  acid,  134 
Amino-polysaccharides,  88 
a-Amino-propionic  acid,  134 
Amino-succinic  acid,  135 
Amino-tyrosine,  test  for,  199 
6-Amino-valerianic  acid,  189 
Ammonia,  output  of,  in  acidosis,  545 
after  parathyroidectomy,  387 
as  source  of  urea,  544 
uric-acid  synthesis  from,  in  birds,  I 

550 
in  urine,  557,  564 

in  acidosis,  276,  576 
Ammonium  carbamate,  545 

carbonate,  transformation  to  urea  in  | 

liver,  544 
formate,  transformation  to  urea  in 

liver,  544 
purpurate,  175 

salts,    permeability    of    blood-cor- 
puscles for,  267 
Amnesia,  523,  531 

Amphibia,  descent  of  birds  from,  262 
Amphibian  blood,  clotting  of,  345 
Amphoteric  acids,  121,  138 

character  of  proteins,  151 
Amygdalin,  76,  89 
Amylase,  228 

in  pancreatic  juice,  229 
Amylin,  86 
Amylodextrin,  86 
Amyloid,  83 

Analysis  of  proteins,  144 
Anaphylactic  shock,  188,  239,  361 

fat-infiltration  of  tissues  in,  286 
methyl-guanidine  in  urine  in, 
194 


Anaphylaxis  after  protein  ingestion,  240 
Anemia,  blood-count  in,  336 

iron  therapy  of,  356 

Anesthetic  action  of  magnesium  salts,310 
Anesthetics,  effects  of,  on  protoplasm,  326 
Animal  fats,  essentiality  of,  in  diet,  585 

proteins,  calorific  value  of,  579 
Annelids,  artificial  parthenogenesis  of,  449 
Antagonistic  salt  action,  318 

origin  of,  321 

Anterior  lobe  of  pituitary,  199,  499,  505 
Antibodies,  239,  331,  600 

enzymes  a^,  226 

in  identification  of  proteins,  224 
Anti-enzymes,  226 

Antigenic  properties  of  compound  pro- 
teins, 332 

proteins,  130 
Antigens,  331 

Antimony  sulphide,  colloidal,  158 
Antipepsin  in  intestinal  worms,  226 
Antiprothrombin,  348 

nature  of,  349 
Antipyrin,  effect  of,  on  globulin-albumin 

ratio,  342 

Antiscorbutics,  193 
Antithrombin,  347 

nature  of,  349 

in  uterine  secretions,  378 
Antitoxins  in  diphtheria,  339 
Antitrypsin  in  intestinal  worms,  226 
Apis  mellifica,  beeswax  from,  112 
Apnea,  366,  367 
Arabinose,  64 

formula,  70 
Arabitol,  64 
Araboric  acid,  64 
Arachnidce,  129 

hemocyanin  in,  350 

Arbacia,  artificial  parthenogenesis  in,  446 
Arenicola,  heliotropism  of,  430 
Arginase,  544 
Arginine,  determination  of,  145 

formula,  135 

relation  of,  to  creatine,  195,  388,  548 

separation  of,  133 

as  urea  precursor,  543 
Ariolimax columbianus,  nerve-conduction 

in,  426 

Aromatic  oxyacids,  558 
Arsenic  as  a  foodstuff,  49 

compounds,  effect  of,  on  metabol- 
ism, 311 

sulphide,  158 
Arteriosclerosis,  237 
Artificial  lipase,  596 

parthenogenesis,  446 

improved  method,  449 
Artificially  fed  infants,  591 
Aspartic  acid,  135 

Aspergillus  oryzce,  diastase  from,  215 
Asphyxia,  366 
Assimilation-limit  of  ammo-acids,  244 

of  carbohydrates,  402 
Association  in  memory,  531 


INDEX  OF  SUBJECTS 


613 


Asthma,  188,  240 
Atophan,  553 

Atropine,  effect  of,  on  pancreatic  secre- 
tion, 373 
in  poisoning  by  choline  and  neurine, 

196 

Atwater-Rosa  calorimeter,  571 
Autocatalysis  in  oxidation  of  linseed  oil, 

111 
Autocatalytic  formula,  528 

application  of,  to  central  ner- 
vous phenomena,  527 
Autocatalyzed  monomolecular  reaction, 

475,  477 
reactions,  439,  524 

curve  of,  475 

Autodestruction  of  enzymes,  425 
Autohydrolysis,  131 
Autolysis,  177 

Aveno  saliva,  heliotropic  curvature,  430 
temperature-coefficient  of, 
428 


B 


BACILLUS  aminophilus  intestinalis,  de- 
carboxylization  of  amino-acids  by, 
186 

cholerce,  relative  permeability  in,  265 
prodigiosus,  proteolytic  enzymes  in, 

206,  214 
subtilis,  antagonistic  salt  effects  of, 

321 
Bacteria,  growth  of,  475,  478,  482 

curves  of,  474 
production  of  nitrogenous  bases  by, 

184 

protein  metabolism  of,  395 
Bacterial   spores,    Arrhenius'  theory  of 

origin  of  life  through,  435 
resistance  of,  282 
Balanced  solutions,  318 
Balanus  eburneus,  calcium  necessary  for 

motility,  325 

Barium  chloride  as  a  purgative,  316 
Basal  metabolism  in  growth,  594 

influence  of  temperature  on,  422 
ratio  of,  to  body  surface,  588, 

590 

in  sea-urchin  eggs,  459 
Basedow's  disease,  386 
Beeswax,  112 

Benzene    administration,  globulin-albu- 
min ratio  in,  341 
membrane-formation  by,  451 
oxidation  of,  in  diabetes,  404 
Benzidine  reaction,  362,  413 
Benzoic  acid  in  urine,  412,  556 
Beri-beri,  191 
Betaines,  189-193 
formula,  189 
Bicarbonates  in  blood,  277 

neutralizing-power  of,  278 
Bile,  231 


r  Bile,  channel  of  sulphur  excretion,  560 
-concretions,  100 
osmotic  pressure  of,  261 
-pigments  in  bile-concretions,  100 

in  urine,  105 
-salts,  102-105,  233 
absorption  of,  237 
as  cholagogues,  105 
as  cytolytic  agents,  451,  453 
effect   of,    on  surface   tension, 

207,  284 
Bilirubin,  563 
Biological  individuality  of  blood,  356 

of  tissues,  330 
values  of  proteins,  584 
Bioluminescence,  414 
Birds,  osmotic  pressure  of  blood-sera,  261 
Birotatiqn,  72 

Birth-weight,  energy  consumed  in  doub- 
ling, 593 

Biuret-reaction,  149 
with  pituitrin,  199 
with  proteins,  123 

Blastula  stage  in  sea-urchin  eggs,  462 
Blood,  absorption  spectrum  of,  350 
alkali  reserve  of,  273,  279 
amino-acids  in,  337 
ammonia  in,  337 
benzidine  reaction  for,  362 
bicarbonates  in,  277 
biological  individuality  of,  356 
chemical  composition  of,  337 

detection  of,  361 
coagulation  of,  342,  348 
composition  of,  335 
cytolytic  power  of  foreign,  453 
defibrinated,  336 
glucose  content  of,  337 
guaiacum  test  for,  362 
hemin  test  for,  362 
phosphates  in,  277 
photographic  spectrum  of,  350 
identification  of  species  of,  362 
platelets,  337,  344 
protein  salts  in,  277 
serum  albumin,  339 
specific  gravity  of,  337 
titratable  alkalinity  of,  273 
urea  in,  337 

Blood-pressure,  effect  of  amines  on,  186 
Blood-serum,  proteins  in,  339 
Bodily  heat,  568 

surface  and  basal  metabolism,  588, 

590 

temperature,  regulation  of,  540 
Boiling  point  of  water,  elevation  of,  by 

dissolved  substances,  259 
Bolina,  426 
Brain,  phospholipine,  114 

ratio  of  weight  of;  to  body  weight, 

517 

Bright's  disease,  96 
i  British  infants,  growth  of,  476 
Bromelin,  214 
a-Brom-iso-capronyl  chloride,  141 


614 


INDEX  OF  SUBJECTS 


a-Bromopropionyl  chloride,  141 

Bronchioles,  ergamine  effects  of,  188 

Brucine,  181 

Buffer-solutions,  279 

Bunsen-Roscoe  law,  430 

Burns,  effect  of,  on  suprarenal  cortex, 
369 

Butter-fat,  409 

Butyric  acid,  108 

in  artificial  parthenogenesis,440 
in  membrane-formation,  451 

7-n-Butyro-bet  aine,  1 90 


CACHBXIA  strumipriva,  382 
Cadaverine,  562 

physiological  action  of,  187 
Cadmium  sulphide,  158 
Caffeine,  176,  563 
Calcium  carbonate  in   bile-concretions, 

100 

chloride  in  blood-coagulation,  342 
inhibition  of  membrane-forma- 
tion, 459 
effect  of  removal  of,  from  tissues, 

312,  314 
excretion,  43 
as  a  foodstuff,  40 
ions  in  blood-coagulation,  342 

in  metabolism,  387 
in  menstrual  fluid,  377 
in  milk-clotting,  249 
in  modified  milk,  42 
precipitants   as   nerve   stimulators, 

374 

in  blood  coagulation,  342 
as  cathartics  and  diuretics,  314 
salts  as  catalyzer  in  adrenaline  test, 

198 

Callianira,  426 
Calorie,  391 
Calorific  requirement   and    surface-law, 

587 

value  of  diet,  582 
values  of  foodstuffs,  569 
Canalization-hypothesis,  524 
Cancer,  515,  601 

death-rate,  581 
Cane-sugar,  76. 

in  artificial  parthenogenesis,  446 
hydrolysis  of,  439 
photosynthesis  of,  437 
toleration  of,  in  cliabetes,  404 
Cannabis  sativa,  edestin  from,  126 
Capillary  electrometer,  443 

forces,  535 

Capric  acid  in  fat  metabolism,  410 
Caprine  formula,  134 
Caproic  acid  in  fat-metabolism,  410 
Caprylic  acid  in  fat-metabolism,  410 
Caramel,  59 
Carbamic  acid,  544 
Carbohydrate  esters,  91 


Carbohydrate  radical  in   nucleic  acids, 

173 

Carbohydrates,  53,  568 
calorific  value  of,  569 
digestion  of,  228 
energy  liberated  by,  396 
intermediate  metabolism  of,  391-399 
oxidation  of,  436 
photosynthesis  of,  434 
source  of  acetone  bodies  in  urine,  405 
Carbon    dioxide,    assimilation    of,     by 

plants,  434 
rate  of,  437 
temperature-coefficient   of, 

429 

determination  of,  in  bloo.d,  279 
as  fatigue-product  in  muscles, 

524 

output,  539 
production  of,   in  germinating 

seeds,  420 

respiratory  control  by,  367,  524 
as  waste  product,  537 
monoxide  hemoglobin,  352 

poisoning,  398 

Carbonaceous  waste  products,  537-541 
Carboxethyl-glycyl-glycine  ester,  140 
Carcinoma,  504 

cholesterol  content  of  tissues  in,  511 
deficiency  of  cholesterol  derivatives 

in,  101 
effect  of  cholesterol  on  growth  of,  99 

of  tethelin,  507 

spontaneous  development  of,  511 
Carica  papaya,  proteolytic  enzyme  in,  214 
Carnaiiba  wax,  1 13 
Carnitine,  395 

formula,  190 
Carnosine,  189 
Cartilage,  effect  of,  on  solubility  of  urates, 

552 

Casein,  129,  157 
anhydrous,  167 
combining  weight  of,  154 
compounds,  164 

conversion  of,  to  paracasein,  345 
effect  of  introduction  of,  into  circu- 
lation, 239 

electrical  conductance    of,  in    solu- 
tions, 157 
as  foodstuff,  492 

formate,  coagulation  and  precipita- 
tion of,  168 
glutamic  acid  in,  136 
in  milk,  591 

separation  of,  in  sour  milk,  40 
rate  of  solution  of,  by  alkali  solu- 
tions, 535 
uses  of,  599 
Castor  oil,  111 
Castration,  376 
Catalase,  414 

Catalysis,  mechanism  of,  203 
Catalyzers  of  growth,  478,  493,  503 
Cataphoresis  in  protein  solutions,  157 


INDEX  OF  SUBJECTS 


615 


Catechol,  197 

-group,  essentiality  in  diet,  480 

oxidation  of,  by  enzymes,  414 
Cathartics,  314 
Cell-contents,  osmotic  pressure  of,  263 

division,  chemical  mechanics  of,  466 

drawings  of,  468 
Cellular  elements  of  blood,  335 
Celluloses,  82-84 

in  diet,  231 
Central  nervous  phenomena,  autocata- 

lytic  nature  of,  527 
Cephalization-f actor,  517 
.  Cerebellar  excitation,  315 
Cerebronic  acid,  91 
Cerebrosides,  91 

galactose  in,  72 

hydrolysis  of,  197 
Ceriodaphnia,  heart-beat  in,  420 
Cerosin,  86 
Cetyl  alcohol,  112 

palmitate,  236 
Chlamydomonas  pisiformis,  heliotropism, 

430 

Chara,  protoplasmic  streaming  in,  445 
Characterization  of  proteins,  144-147 
Chauvenet's  criterion,  432 
Chemical  mechanics  of  cell- division,  466 

of  muscular  contraction,  438 
Children,  nutrition  of,  590 
Chinese  wax,  1 12 
Chitin,  88 
Chloracetyl  glycyl-glycine  ester,  140 

group  in  polypeptide  synthesis,  141 
Chloral  hydrate,  effect  of,  on  fertilized 

eggs,  460 

on  pancreatic  secretion,  3  74 
narcosis,  294 

Chlorine  in  foodstuffs,  49 
Chloroform,  effect  of,  on  globulin-albu- 
min ratio,  342 

emulsions,  288 

membrane-formation,  451 

-poisoning,     fibrinogen-content     of 

blood  in,  348 
Chlorophyll,  47,  429 

carbon-dioxide   assimilation  of,  434 

fate  of,  in  alimentary  canal,  356 

formaldehyde  synthesis  in  vitro,  436 

relationship  of,  to  hematin,  355 
Chloroplasts,  photosynthesis  of,  436 
Chlorosis,  43,  601 
Cholagogues,  105 
Choleic  acid,  104 
Cholesterol,  90 

absorption  of,  237 

in  bile-concretions,  100 

crystal-form,  98 

in  diabetic  blood,  406,  409 

in  diet,  94,  492 

effect   of,    on  development   of  sea- 
urchin  eggs,  465 
on  growth  of  carcinoma,   504, 

511 
on  growth  of  mice,  505 


Cholesterol  esters,  101 

saponification  of,  by  lipase,  237 
in  suprarenal  cortex,  369 
formula,  97 

as  a  growth  catalyzer,  500 
Cholic  acid,  93,  102-104 
i  Choline  in  cell  division,  468 
formula,  etc.,  196 
in  nuclear  synthesis,  469 
oleate,  468 
Chondroitin,  92,  129 

sulphuric  acid,  91,  562 

in  chondroproteins,  129 
Chondroproteins,  129 
Chromoproteins,  129 
Chyme,  228 

discharge  of,  into  intestine,  372 
Chymosin,  249 

Cinchonine,  effect  of,   on  creatine  con- 
tent of  muscle,  399 
Cipollina's  test,  68 
Circulatory  system,  chemical  regulation 

of,  368-371 

I  Clemmys  marmorata,  temperature  coeffi- 
cient of  heart  beat,  420 
Clotting  of  bird  and  amphibian  blood, 

345 

of  blood,  342 
effect   of,    on    heat   production   in 

infants,  591 
Clupeine,  170 
Coagulated  proteins,  131 
Coagulating  agents,   effect   of,   on  per- 
meability, 295 
Coagulation,  131 

of  blood,  337,  342,  348 
effect  of,  on  structure,  300 
of  proteins,  121,  158,  164 
reactions,  121-122 
!  Coagulative  power  of  salts,  164 
Cobalt  chloride,  166 
!  Cod-liver  oil,  110 

assimilation  of,  236 
Cold  light,  596 
Collagen,  127 

Collodion  gel,  sponge  structure  of,  298 
!  Colloids,  precipitation,  158 
i  Color  blindness,  348 

reactions  of  proteins,  122 
Colostrum,  381 
Columnar  epithelium,  290 
Conduction  of  stimuli,  426 
Conductivity,  effect  of  temperature  on, 

418 

Conjugated  excreta,  554-558 
glucuronic  acids,  554 
proteins,  128-130 
sulphates,  559 
Connective  tissue  in  growth,  510 

.proteins  of,  245 
I  Conservation  of  energy,  567,  571 

of  matter,  law  of,  32,  567 
Copper  ferrocyanide  membranes,  257 
Coprosterol,  98 
Corpora  lutea,  377,  378,  493 


616 


INDEX  OF  SUBJECTS 


( lotion-seed  oil,  111 
Crcatine,  194,  388,  395,  515 

estimation  of,  548 

formula,  388 

muscle  content  of,  in  work,  399 

in  muscles  of  various  animals,  194 

stimulation  of  nerve  cells  by,  428 

in  urine,  547 
Creatinine,  195,  545-547 

formula,  545 

output  of,  in  inosite  administration, 
96 

tests  for,  548 

Creosote,  membrane  formation  by,  451 
Cresol  sulphuric  acid  in  urine,  556 
Cretinism,  382,  601 
Cruciferce,  glucosides  in,  89 
Crustacea,  chitin  in,  89 

hemqcyanin  in,  129,  350 
Cryoscopic  method,  260 
Crystal  habit,  357 
Crystalline  serum  albumin,  339 
Ctenophores,  426 
Cuorin,  116,  349 

Cupric  bromide,  dehydration  of,  by  salts, 
166 

caseinate,  167 

chloride,  dehydration  of,  166 

effect   of,    on    permeability   of 

paramecium,  295 
Curare,  194,  312 

effect  of,  on  lymph  flow,  363 
Curve  of  forgetting,  535 

of   growth,    carcinoma,  in   tethelin 

rats,  507 

in  cholesterol-fed  mice,  505 
in  tethelin-fed  mice,  506 
Cyanides,  effect  of,  on  cell  oxidations,460 
on  central  nervous  system,  501 
Cyclamen,  glucosides  in,  90 
Cyclodus  gigas,  carbon  dioxide  output, 

540 

Cycloses,  95-96 
Cypridina,  photogenin  in,  414 
Cystine,  561-562 

detoxication  of  ultraviolet  light  by, 
433 

essentiality  for  tissue  accretion,  490 

estimation  of,  145 

formula,  134 

in  keratin,  127 

separation  of,  133 

test  for,  562 

in  urine,  562 
Cystinuria,  562 
Cytidine,  178 
Cytolysis,  451,  458 

in  sea  urchin  egg,  452 
Cytolytic  agents  in  luminescence,  415 
Cytosine,  174-175 


DEAMINIZATION,  184,  244 

effect  of  thyroid  secretion,  385,  486 


Dcaminized  gelatin,  155 

proteins,  124 
Death-rate,  581 
Decarboxylization,  373 

by  bacteria  and  fungi,  184 

•  Decomposition  products  of  nucleic  acids. 

173 

Defibrinated  blood,  336 
Dehydration  in  protein  coagulation,  158, 

165,  167 
Dendrites,  522 

*  Dendrostoma,  blood  of,  as  cytolytic  agent, 

463 

i  Desoxycholic  acid,  104 
Deuteroproteose,  130 
Development,  effect  of  temperature  on, 

426 

reversal  of,  460 
Dextrins,  82 

i  Dextrose-nitrogen  ratio,  403 
Diabetes,  399-412,  601 
acidosis  in,  276 
blood-fat  in,  236 
fat  tolerance  in,  576 
inosite  in  urine,  96 
insipidus,  563 
mellitus,  399 
spontaneous,  402 
;  Diabetic  coma,  405,  408 

puncture,  400 
Diacetyl,  195 

Diamines,  physiological  action  of,  187 
|  Diamino  acids,  389 

carboxylic  acids,  133 
hydroxy-monocarboxylic  acids,  133 
mpnophosphatids,  114 
trioxydodecanic  acids,  133 
a-to-Diamino  caproic  acid,  135 
|  Diastase,  78,  206,  215 

synthetic  action  of,  223 
Diazobenzene  sulphonic  acid,  199 
oi-5-Dibromovaleryl  chloride,  141 
Dichroism,  351 

in  hematin  solution,  354 
Dicystein,  134 
Diet,  essential  constituents  of,  488 

normal,  582 
Dietary,  effect  of,  on  bodily  dimensions, 

485 

fads,  51 

Diffusion,  effect  of  temperature  on,  418" 
Digestion,  377 

and  absorption,  time  and  mass  rela- 
tions, 250-252 
of  carbohydrates,  228-232 
of  fats,  232-237 
of  proteins,  238-249 
Digitalis  purpurea,  glucosides  in,  89,  90 
j  Digitonin,  90 
j  Diglycerides,  107 
i  Diglycyl-glycine,  141 
i  Dihydrophenols,  199 
Diketo-piperazine,  143,  169 
Dilaurate  of  isomannitol,  235 
Dimethyl-guanidine,  388,  395 


INDEX  OF  SUBJECTS 


617 


Dipeptides,  132,  142 

Diphtheria,  339 

antitoxin,  serum  sickness,  240 

Disaccharides,  53,  76-81 

enzymatic  hydrolysis  of,  218 

Distilled  water  as  cytolytic  agent,  451 

Diurates,  549 

Diuresis,  caused  by  adrenaline  injection, 

370 
by  calcium  precipitants,  314 

Diuretics,  563 

Dolphins,  spermaceti  from,  112 

Drying  oils,  autocatalysis  in,  440 

Ductless  glands,  376 

Dulcitol,  63 

Duodenum,  reaction  of  contents  of,  374 

Duration  of  life,  518 

effect  of  tethelin  on,  516 
temperature  coefficient  of,  424 

Dysmenorrhea,  378 

Dyspituitarism,  498 

Dyspnea,  277,  366 

Dystrophia-adiposo-genitalis,  497 


ECK'S  fistula,  542 
Eclampsia,  395 

creatinine  output  in,  195 
Edema,  308 
Edestin,  126,  150,  152 

copper  compounds  of,  155 
effect  of  introduction  of,  into  circu- 
lation, 239 

formation  of  acid  by,  in  solution,  328 
Efficiency  of  surface  tension  engine,  441 
Egg  albumin,  143,  159 

digestion  of,  by  pepsin,  212 

electrolyte  free,  164 

osmotic  pressure  of  solution  of, 

303 

precipitation  of,   by  silver  ni- 
trate, 164 

recrystallization  of,  361 
Eggs,  hypersensitivity  to  proteins  of,  240 
osmotic  pressure  of  contents  of,  261 
in  vegetarian  diet,  585 
Ehrlich's  reaction,  69,  117 
Elasmobranchii,     osmotic     pressure     of 

serum,  262 

Elastic  tissue,  composition  of,  245 
Elasticity  of  cell  membranes,  264 
Elastin,  121,  127,  143 
properties  of,  599 

Electrical  conductivity  of  blood,  336 
of  protein  solutions,  296 
of  sea  urchin  eggs,  294 
potential  changes  in  muscular  con- 
traction, 443 

sign  of  ions  in  precipitation  of  col- 
loids, 158 

Electrolyte-free  albumin,  164 
Electrolytic  dissociation,  260 
Electronegative  colloids,  159 


Electronegative  protein,  159 
Electropositive  protein,  159 
Electrostatic  tension  in  gelatin  swelling, 

306 

Elodea  canadensis,  toxicity  of  formalde- 
hyde for,  435 

Embryonic  tissues,  water  content  of,  502 
Emotional  glycosuria,  371 

states,  effect  of,  on  suprarenals,  371" 
Emphysema    on    ergamine    administra- 
tion, 188 

Emulsification  of  fat  in  normal  cells,  286 
by  pancreatic  juice,  233 
in  water,  286 
Emulsin,  78,  89,  222 
Emulsion,  fat,  107 

structure  of  protoplasm,  284 
water  in  oil,  287 
Emydura  macquarice,  osmotic  pressure  of 

serum,  262 
Emys  europea,  osmotic  pressure  of  blood 

serum,  262 
temperature    coefficient    of    heart 

beat,  420 
Endocrine  organs,  376 

relationship  of,  to  growth,  493 
Endogenous  catalyzers,  478,  482,   502, 

503,  509,  589,  590 
metabolism,  482,  490 
creatinine  in,  546 
of  purines,  549 
of  sulphur,  560 

End-product  of  hydrolysis,  131 
Energy  equivalent  of  growth,  592 

transformations  in  living  organisms, 

417-445 

Enterokinase,  346,  375 
Environment,  fitness  of,  282 

influence  of,  on  growth,  484-488 
Enzymatic  hydrolyses,  influence  of  reac- 
tion on,  217 

Enzyme  reactions,   temperature   coeffi- 
cient of,  425 
Enzymes,  201-226 

autodestruction  of,  418 
inactivation  of,  216 
influence  of  temperature  on,  213-217 
oxidizing,  412-414 
quantitative  relationships  of,  207 
reaction,  influence  of,  217-218 
specificity  of,  218 
synthesis  by,  221 
thermolability  of,  346 
Eosin,  inactivation  of  enzymes  by,  216 

as  photochemical  sensitizer,  432 
Epinephrin,  197 
Epithelial  cells,  radial  symmetry  in,  292 

tissues,  growth  of,  508 
Equilibria    in   thermodynamical    equa- 
tions, 265 

Equilibrium,  503,  513 
constant,  353 

effect  of  temperature  on,  417 
Equisetum,  salt  antagonism  in,  320 
Erepsin,  184,  238,  248,  375 


618 


INDEX  OP  SUBJECTS 


Ergamine,  187 

relation  of,  to  pituitary  extract,  199 
Ergot,  187 
Ergotoxine,  187 
Erucic  acid,  235 
Erythritol,  267 
Erythrocytes,  335 
Ether,    effect    of,    on    globulin-albumin 

ratio,  342 

Ethereal  sulphates,  559 
Ethyl  acetate,  hydrolysis  of,  211 

membrane  formation  by,  448 
alcohol,    effect    of,    on    pancreatic 

secretion,  374 
butyrate,    enzymatic   synthesis    of, 

223 

hydrolysis  of,  221 
Ethylene  glycol,  permeability  of  blood 

corpuscles  for,  268 
Eudendrium,  heliotropism,  430-43 1 
Euglena  viridis,  heliotropism,  430 
Euglobulin,  339 
Exercise,  effect  of,  on  heat  production; 

591 

Exogenous  metabolism,  482,  485,  494 
in  growth,  592 
of  proteins,  580 
of  purines,  549 
of  sulphates,  560 
thyroid  control,  581 
urea  in,  546 

Exophthalmic  goiter,  386 
External  phase  of  gels,  300 


F 


FATIGUE  in  memory,  521 
muscular,  398,  439 
products  of  nerve  centers,  524 

in  sleep,  530,  532 
Fat  soluble  A,  489 

solvents  as  cytolytic  agents,  451 

effect  of,  on  cells,  294 
Fats,  107-111,  568 

absorption  of,  232,  250 
calorific  value  of,  569 
carbohydrates  and,  isodynamic  val- 
ues of,  575 
digestion  of,  232-236 
effect  of  exclusion  of,  from  diet,  403 

on  pancreatic  secretion,  373 
emulsification  of,  in  digestion,  233 
essentiality  in  diet,  489 
in  genesis  of  acidosis,  405 
intermediate  metabolism  of,  399 
respiratory  quotient  of,  538 
as  source  of  muscular  energy,  396 
Fatty  acids,  108 

effect  of,  on  surface  tension,  284 
in  olive  oil,  467 

Fehling's  method  of  sugar  estimation,  58 
solution,  action  of,  mode  of,  413 
on  nucleosides,  178 


Ferric  chloride  reaction  for  aceto-acetic 

acid,  405 

for  adrenaline,  198 
Fertilization,  446 
membrane,  447 

causative  agents  of,  294,  450 
Fever,  temperature  coefficient  of  heart- 
beat in,  422 
i  Fibrils,  441 
Fibrin,  335,  343 

chemical  nature  of,  348 
crystals,  349 
Fibrinogen,  336,  343 

action  of  thrombin  on,  345 
chemical  nature  of,  348 
migration  of,  toward  electrodes,  157 
source  of,  348 
Fibroblasts,  509 
Fibroin,  121,  127,  143 
properties  of,  599 

Fibrous  tissues,  glycocoll  content  of,  245 
Field  of  consciousness,  531 
Fischer's  amino-acid  method,  results  of, 

146 
Fishes,  osmotic  pressure  of  blood  serum 

of,  262 

Fistuke  in  digestion  studies,  241 
Fluid  crystals,  102 
Fluorine  as  foodstuff,  49 
Foaming  in  protein  solutions,  prevention 

,of,  274 
Foods,  definition  of,  33 

requirement  in  infants,  591 
Foodstuffs,  485 

classification  of,  33 
in  growth,  471,  482 
as  substrates  of  growth,  488 
Forgetting,  velocity  of,  533 
Formaldehyde,  action  of,  on  casein,  599 

in  carbohydrate  synthesis,  435 
Formic  acid,  108,  174 

in  anaerobic  decarboxylization, 

184 

solution  of  casein  in,  167 
Formol  titration,  564 
Free  amino-groups,  149 

in  unhydrolyzed  proteins,  150 
carboxyl-groups  in  proteins,  153 
Martin,  377 

Freezing-point,    lowering    of,     by    dis- 
solved substances,  259-260 
Frohlich's  disease,  498 
Fructose,  57 

photosynthesis  of,  437 
d-Fructose,  77 
Fucus,  artificial  parthenogenesis  in,  450 

as  source  of  polysaccharides,  84 
Fundulus,  eggs,  development  in  balanced 

solutions,  318 

formation  of  monstrosities,  470 
temperature  coefficient  of  heart-beat, 

421 
Fungi,  phytosterols  in,  100 

production  of  nitrogenous  bases  by, 
184 


INDEX  OF  SUBJECTS 


619 


Funnel-shaped  pores,  292 
Furfurol,  62 


G 


GALACTIN,  86 
Galactose,  79-80,  230 
in  diabetes,  404 

distribution  in  animal  kingdom,  72 
d-Galactose,  79,  230 
Galanthus  nivalis,  photosynthesis  in,  437 
Gallois's  reaction,  96 
Ganassini's  reaction,  549 
Gas  chain,  154 
Gastric  digestion,  248,  251 
juice,  amylase  in,  228 
lactase  in,  228 
lipolytic  action  of,  232 
quantity  excreted,  250 
secretion  of,  371 
Gelatin,  127,  143 

in  chloroform  emulsions,  288 
dietary  deficiencies  of,  577 
as  foodstuff,  491 

gels,  velocity  of  diffusion  of  crystal- 
loids through,  296 
structure  of,  299 

effect  of  coagulation    on, 

300 

glycine  content  of,  136 
indican  output  on  diet  of,  555 
liquefaction  of,  by  trypsin,  213 
properties  of,  599 
as  a  protein  sparer,  491 
swelling  of,  305 
Gelatinization,  298 
Generative  organs,  chemical  correlation 

of,  376-380 

Gestation,  period  of,  380 
Gigantism,  494 

tethelin  in,  118 
Gliadin,  127,  156 

amino-groups  lacking  in,  490 
dietary  deficiencies  of,  577 
digestion  of,  241 
peptides  in  hydrolysis  of,  143 
selective  absorption  of  ammo-acids 

of,  246 
Globin,  129,  350 

caseinate,    antigenic   properties   of, 

332 

compound  with  casein,  170 
ultraviolet  spectrum  of,  351 
Globulin-albumin  ratio  in  blood,  339,  340 
Globulins,  126,  136,  159 
Glucohemia,  79,  229,  399,  402 
in  diabetic  puncture,  400 
following   intravenous   injection   of 

adrenaline,  370 
Gluconic  acid,  404 
Glucoproteins,  129 
Glucosamine,  66 

in  chondroitin,  129 
formula  for,  69 


Glucose,  86 

in  blood,  337 

formation  of,   from  propionic  acid 
in  tissues,  411 

identification  of,  in  urine,  402 

photosynthesis  of,  437 

in  urine,  229 
a-5-Glucose,  74,  238 
0-5-Glucose,  74 
5-Glucose,  71,  77,  230 
Glucose-glycogen  equilibrium,  400 
Glucosides,  76,  89-91 

analogy  of,  to  nucleosides,  178 

enzymatic  hydrolysis  of,  218 
Glucothionic  acid,  91 
Glucuronates,  554 
Glucuronic  acid,  62,  66-67 
in  chondroitin,  129 
formula,  404 
naphtho-resorcinol  reaction  of, 

67 

origin  of,  in  urine,  555 
Glutamic  acid,  135 
Glutelins,  126 
Glutenin,  126 

Glycerol,  permeability  of  blood  corpus- 
cles for,  268 

solutions,  viscosity  of,  296 
Glycerophosphoric  acid,  237 
Glycerose,  53 
Glycine,  anhydride,  139 

crystal  form,  137 

formula,  134 
Glycocholic  acid,  237 
Glycocoll,  from  hydrolysis  of  glycocholic 
apid,  103 

in  fibrous  tissues,  245 

synthesis  of,  by  living  tissues,  490 

in  urine,  554 

as  vehicle  of  excretion,  556 
Glycogen,  71,  82,  86-88,  229,  230 

content  of  heart,  501 

distribution  of,  in  body,  88 

in  muscular  work,  391 

synthesis  of,  by  diastase,  223 
Glycogen-glucose  equilibrium,  400 
Glycoleucine,  134 
Glycosuria,  70,  79,  399 

adrenaline,  370 

alimentary,  72 

emotional,  371 

from  extirpation  of  mammary 
glands,  230 

from  magnesium  chloride  in  blood, 
318 

phloridzin,  398 

sea-water,  271 
Glycyl-glycine,  140,  156 

chloride,  139 
Goiter,  386 

Gonionenus,  rhythmic  contractions  in,  3 13 
Gout,  601 

uric  acid  in,  552 
Graafian  follicle,  377 
Grape  sugar,  71 


620 


INDEX  OF  SUBJECTS 


Gray  matter,  insensitivity  of,  to  calcium 

precipitants,  315 
Growth  catalyzers,  503 

effect  of,  on  parenchyma,  516 
curve  in  female  white  mice,  512 

in   normal   and   pituitary   fed 

mice,  499 
cycles,  472-475 
effect  of  cholesterol  on,  98 
energy  equivalent  of,  592 
guinea-pig,  man,  tumors,  472 
impeding  factors  in,  478 
process  of,  general  characteristics  of, 

471-484 
influence  of  race  and  sex  on, 

484-488 

regenerative,  472 
relationship  of  endocrine  organs  in, 

493-500 

substrates,  483-493 
Guaiaconic  acid,  362 
Guaiacum  test,  413 

tincture,  412 
Guanase,  177 
Guanidine,  177,  194 

tetany,  389 
Guanine,  174,  176 

from  thymus  nucleic  acid,  389 
Guanosine,  178-180 
Guanylic  acid,  174,  178,  180 
Guinea-pig,  growth  of,  472 
Gums,  82,  599 


HAMMARSTEN'S  reaction,  103-104 
Heart  beat,  temperature  coefficient  of, 
v     420 

ganglion,  temperature  effects  on,  421 
in  inanition,  501 

Heat  coagulation  of  proteins,  122 
of  combustion,  443,  569 
evolution,  Lavoisier's  work  on,  568 
production  in  infants,  591 

in  resting  animals,  588 
Heavy  metals,  oligodynamic  action  of 

328,  330 
salts,  effect  of,  on  protoplasm, 

311 

in  protein  precipitation,  122 
Hehner,  number  of  fats,  110 
Heliotropism,  429-432 
Heller's  test,  122 
Hematin,  126,  129,  350,  354,  355 
Hematocrit,  266 
Hematogen,  45 
Hematoidin,  355 
Hematoporphyrin,  45,  355 
Hemin,  crystals,  etc.,  354-355 

test,  362 

Hemochromogen,  355 
Hemocyanin,  129,  350 
Hemoglobin,  43,  129,  343 

absorption  spectrum  of,  351 


Hemoglobin,  casemate,  170 
chemistry  of,  350 
coagulation  of,  temperature  effects 

of,  426 

compounds  with  oxygen,  352 
crystal  form,  356 
crystals,  how  obtained,  356 
iron  content  of,  350 
in  luminescence  of  pyrogallol,  415 
migration  of,  in  electric  field,  157 
molecular  weight  of,  353 
in  nutrition,  488 

osmotic  pressure  of,  302,  303,  353 
oxygen  saturation  of,  in  respiration, 

367 

as  oxygenase,  413 
recrystallization  of,  361 
Hemolysins,  331 
Hemplysis,  90,  266 

inhibition  of,  by  proteins,  456 
Hemolytic  agents,  membrane  formation 

by,  451 

Hemophilia,  347 
Hemopyrrole,  355 
Hempseed,  edestin  from,  126 
Hensen's  line,  442 
Hepatic  cirrhosis,  348 
Hexoses,  53,  55,  62,  174 
Hexylamine,    blood-pressure   effects   of, 

186 

Hibernating  animals,    respiratory   quo- 
tient of,  539 
Hippuric  acid,  synthesis  of,  in  kidney 

tissue,  32 
in  urine,  412,  556 
Hirudin,  343,  349 
Histidine,  66,  188,  189 
crystal  form  of,  137 
determination  of,  145 
formula,  135 
separation  of,  133 
Histones,  126,  136 
Homarus,   composition  of    blood  serum 

of,  269 

Homogentisic  acid,  558 
Homoptera,  wax  production  by,  112 
Honeycomb  structure  of  gels,  300 
Hopkins-Cole  reaction,  122,  124 
Hordein,  127 
Hormones,  335 
Horse  serum,  crystalline  serum  albumin 

from,  339 
Human  growth,  473,  475 

milk,  composition  of,  590,  591 
Humin,  82 

substances,  62,  66 

relation  of,  to  iodothyrin,  384 
Hyaloplasm,  442 
Hydremic  plethora,  363 
Hydrochloric  acid  of  gastric  juice,  228, 

328 

membrane  formation  by,  451 
specificity  of,  in  pepsin  hydro- 

lyses,  217 
Hydrocyanic  acid,  action  of,  on  aldoses,65 


INDEX  OF  SUBJECTS 


621 


Hydrogen  electrode,  154,  273 

ions,  as  cytolytic  agent,  452 

in  membrane  formation,  450 

peroxide,  362,  412 
Hydrolysis,  131 

in  development  of  tissue,  460 

in  living  tissue,  365 

of  proteins,  products  of,  130,  155 

temperature  effect  on,  417 

velocity  of,  201-223 
Hydrolyzing  enzymes,  201-226 

synthetic  action  of,  221 
/3-Hydroxy-a-amino  propionic  acid,  134 
Hydroxyaromatic  derivatives,  metabol- 
ism of,  in  diabetes,  409 
Hydroxybutyric  acid  in  diabetic  urine, 

405 

in  oxidation  of  fats,  408 
Hydroxyhexahydrophthalic  acid,  104 
Hydroxyl  ions,  concentration  of,  273 

as  cytolytic  agents,  452 
a-Hydroxypyridine,   192 
Hydroxy-a-pyrrolidine   carboxylic   acid, 

135 

Hymenoptera,  wax  production  by,  1 12 
Hypaphorine,  190 

Hyperpituitarism,  glycosuria  in,  400 
Hyperpriea,  366 
Hyperthyroidism,  386 
Hypertonic  sea  water,  461 

in  artificial  parthenogene- 
sis, 446,  449 

solution,  264,  266 
Hypnosis,  522 

definition  of,  531 
Hypnotoxin,  532 
Hypophysectomy,  498 
Hypopituitarism,  sugar  tolerance  in,  400 
Hypotheses  in  science,  22-44 
Hypotonic  solutions,  266,  459 
Hypoxanthine,  176,  177,  179 

enzymatic  oxidation  of,  412 

formula,  549 

in  oocytin,  455 
Hysteresis  in  reversible  gels,  299 


ICTERUS,  105 
Identical  twins,  469 
Iminazole  ring,  66 

Iminazolyl  group,  essentiality  of,  in  diet, 
489 

radical,  176 

in  tethelin,  117 

/3-Iminazolyl-a-aminopropionic  acid,  135 
jS-Iminazolylethylamine,  381 

in  intestinal  mucosa,  373 
Immunity,  188 
Inactivation  of  enzymes,  216 
Incubation,  osmotic  pressure  of  eggs  dur- 
ing, 261 

India  rubber,  inosite  in  leaves  of,  96 
sponge  structure  of,  298 


Indican,  554 
Indicator  method,  275 
Indigo  from  indican,  555 
Indole,  124,  186 

group,  essentiality  of,  in  diet,  489 
jS-Indole-a-aminopropionic  acid,   135 
Indolethylamine,  physiological  action  of, 

187 

Indoxyl,  67 

Indoxylglucuronic  acid,  67 
Infantile  growth  cycle,  475 
Infants,  extra-uterine  growth  of,  476 
Infraproteins,  130 

synthesis  of,  224 
Infundibulum  of  pituitary,  199 
Inhibitive  factor  in  growth,  480 
Inorganic  environment,  310-327 

salts,  action  of, .on  protoplasm,  311 
precipitation  and  coagulation  of 
proteins  of,   158 

sulphates,  559 
Inosinic  acid,  174,  178,  179 
Inosite,  93,  95,  117 

oxidation  of,  in  diabetes,  404 
Insecta,  chitin  in  exoskeleton,  88 

rhythmic  contraction    of,   in  intes- 
tines, 313 

Insoluble  serum  globulin,  339 
Intermediate    metabolism    of    carbohy- 
drates, 391-399 
of  fats,  399-412 
Internal  phase  of  a  gel,  300 

secretion,  489 

work,  muscle  tonus  in,  392 
Interstitial  cells  of  testes,  376 
Intestine,  absorption  of,  250 
Intestinal  epithelium,  one-sided  permea- 
bility of,  291 
selective  absorption  by,  246 

putrefaction,  560,  563 
on  flesh  diet,  586 

stasis,  555 

worms,  anti-enzymes  in  tissues  of, 

226 

Intravascular  clotting,  343 
Inulin,  71,  82,  86 

in  diabetes,  404 
Inulinase,  86 
Inversion  of  sugar,  77 
Invertase,  78,  204,  207,  215 

absence  of,  from  digestive  juice,  229 

specificity  of,  219 

time  relations  in  action  of,  209 
Iodine  in  marine  algse,  329 

number  of  fats,  110 

reaction  for  adrenaline,  198 

in  thyroid,  50,  329,  384 
lodothyrin,  384 

lonization  of  proteins,  157,  296 
Iron  in  anemia,  44 

content  of  foods,  47 

as  foodstuff,  43 

in  nucleoproteins,  129 

in  oxidizing  enzymes,  413 
Irreversible  coagulation,  131 


622 


INDEX  OF  SUBJECTS 


Islets,  of  Langerhans,  401 

Isobutylamine,  effect  of,  on  blood-pres- 
sure, 186 

Isodynamic  foodstuffs,  396 
values  of,  575 

Isolactose,  76,  80 
synthesis  of,  223 

Isoleucine,  134 

Isomaltose,  76,  79,  222 

Isomannitol  dilaurate,  absorption  of,  235 

Isomorphism  in  hemoglobin  crystals,  357 

Isomorphous  salts,  physiological  action 
of,  310 

Isotonic  solutions,  263 


JAFFE'S  reaction,  195,  548 
Japan  wax,  112 
Jaundice,  105 
Jecorin,  117,  349 
Jellies,  298 


KEPHALIN,  116,  344 

in  blood  coagulation,  345 

saponification  of,  196 

thromplastic  powers  of,  346 
Kephir  yeast,  79,  223 
Kerasin,  91 
Keratin,  121,  127,  509 

composition  of,  245 

cystine  content  of,  136 

properties  of,  599 
Ketone  structure  of  sugars,  58 
Ketoses,  62 

Kidney,  development  of,  in  protoverte- 
brates,  270 

one-sided  permeability  of,  291 
Krause's  membranes,  442 


LACCASE,  346,  412 

function  o/  manganese  in,  413 
synthetic,  413 
Lactalbumin,  492 
Lactase,  78,  79,  223 

absence  of,  from  digestive  juices,  229 
in  gastric  juice  of  calf,  228 
Lactic  acid,  397 

in  carbohydrate  oxidation,  438 
effect  of,  on  peptic  hydrolysis, 

217 
fatigue  product  of,  in  muscle, 

217 

in  muscle  fatigue,  439 
oxidation  of,  in  diabetes,  404 
of  fats  and  sugars,  399 
of  glycogen,  397 
in  respiratory  control,  524 


Lactic  acid  in  urine,  541 

of  hibernating  animals,  539 
(8-Lactic  acid,  411 
Lactone  structure  of  sugars,  72-74 
Lactose,  72,  76,  79,  223 

in  diabetes,  404 

possible  forms  of,  80 
Laminaria,  Osterhout's  experiments  with, 

323 
Lanoline,  101 

non-absorption  of,  235,  237 
Large  intestine,  absorption  from,  252 
Latent  period  in  muscle  stimulation,  428 
Laurie  acid,  235 
Lavosin,  86 
Law  of  conservation  of  energy,  567-575 

of  mass  action,  203 

Least  squares,  method  of,  in  biochemis- 
try, 26 
Lecithin,  90,  115 

absorption  and  digestion  of,  237 

distribution  of,  in  cell,  284,  286 

formula,  113 

as  growth  catalyzer,  500 

in  nuclear  synthesis,  463,  469 

retardation  of  development  by,  464 

saponification  of,  196 

source  of  methylamine  in  putrefac- 
tion, 185 

Lecithoproteins,  129 
Leucine,  action  of  ultraviolet  light  on, 
433 

crystal  form  of,  137 

formula,  134 

Leucyl-diglycyl-glycine,  142 
Leucyl-glycyl-glycine,  141 
Leukocytes,  335 
Leukocytosis,  341 
Levulinic  acid,  62,  174 
Levulose,  77,  230 

in  diabetes,  404 

distribution  of,  71 

in  urine,  229 
Lichenin,  86 
Lichens,  86 

Lieberman-Burchard  reaction,  99 
Liesegang  rings,  301 
Life  duration,  effect  of  temperature  on, 

426 

relation  of,  to  cephalization  fac- 
tor, 517 

processes,  influence  of  light  on,  429 

of  temperature  on,  417 
Lifschutz's  reaction,  100 
Light,  influence  of,  on  life  processes,  429 
Lignification,  84 
Lignin,  84 
Lignoceric  acid,  91 

Limulus,  composition  of  blood  serum  of, 
269 

effect  of  temperature  on  heart  gang- 
lion of,  421 
i  Linseed  oil,  111 
JLipase,  107,  115,  234 

in  gastric  juice,  232 


INDEX  OF  SUBJECTS 


623 


Lipase  in  pancreatic  juice,  233 

specificity  of,  219 

synthetic  action  of,  223 
Lipemia,  237 

in  diabetes,  406 

Lipman's  capillary  electrometer,  443 
Lipoid  theory  of  narcosis,  294 
Lipoids,  113 

distribution  of,  in  cell,  286 

non-antigenic,  332 

in  suprarenal  cortex,  369 

surface  tension  and,  284 
Lithium  as  foodstuff,  49 

urate,  553 
Liver,  deaminization  in,  244 

extirpation  of,  effect  on  uric  acid 
excretion,  543 

oxidizing  enzymes  of,  412 

as  source  of  fibrinogen,  348 

urea  formation  in,  542-543 
Living  matter,  methods  of  study  of,  19 
Local  anesthetics,  effect  of  adrenaline  on, 

370 

Lock  and  key  hypothesis,  219 
Locke's  solution,  268 
Longevity,  518 
Lucerne,  laccase  from,  412 
Lupinus  luteus,  galactin  in,  86 
Lymph  as  distributing  agent,  335 

flow,  363 

origin  and  composition  of,  362 

osmotic  pressure  of,  261 
Lymphagogues,  363 
Lysine,  562 

determination  of,  145 

deficiency  of,  491 

essentiality  of,  for  tissue  accretion, 
490 

formula,  135 

nitrogen,  150 

separation  of,  133 


M 


MAGNESIUM  as  foodstuff,  49 

formation  of  monstrosities,  470 

purgative  action  of,  317 

in  tissue  fluids,  269 

salts,  anesthetic  action  of,  310 

glycosuria  in,  399 
Maintenance  metabolism,  594 
Malignant  tumors,  growth  of,  472 
Maltase,  78 

in  pancreatic  juice,  229 
Maltose,  76,  78,  86      . 
in  photosynthesis,  437 
possible  forms  of,  80 
synthesis  of,  by  emulsin,  222 
tolerance  of,  in  diabetes,  404 
Mammary  glands  in  castrated  animals, 

377 

effect  of  pituitary  extract  on, 
199 


Mammary  glands,  relation  of,  to  growth 

of  embryo,  379 

Mandelic  acid  ester,  hydrolysis  of,  219 
Manganese  in  laccase,  412 
Mannitol,  63 

oxidation  of,  in  diabetes,  404 
Mannosaccharic  acid,  64 
Mannose,  438 

Manometer  for  osmotic  pressure  meas- 
urement, 258 
Mass  law,  162,  203 
Master  reaction,  478 

in  food  assimilation,  48 

in  growth  process,  483 

Meat  consumed  by  different  countries. 

579 

Mechanical  work,  574,  576 
Medicago  saliva,  laccase  of,  346,  413 
Medicine,  relation  of,  to  biochemistry, 

600 
Medulla  oblongata,  respiratory  control 

by,  365 
Melanins,  413 
Melibiose,  81 
Membrane  formation,  448,  450,  458 

effect  of,  on  cell  oxidations,  461 
Memory,  521-524 

time  relations  of,  529 
Memory  trace,  526,  530 

fading  of,  532 
Menstrual  fluid,  377 
Mercapturic  acid,  562 
Mercuric  chloride,  adrenaline  test  of,  198 
Metabolic  rate,  500,  501,  509,  589 
of  infants,  591 
of  nervous  tissues,  517 
in  old  age,  512 
Metabolism,  basal,  422,  459,  588,  590, 

594 

carbohydrate,  391 
chemical  regulation  of,  381-389 
of  children,  592 
fat,  399 

intermediate,  391,  399 
maintenance,  594 
stimulation,  of,  by  exertion,  589 
Metamorphosis,  499 

influence  of  thyroid  on,  486 
Metastases    in     carcinoma,    cholesterol 

effect  of,  504 
Metazoa,  growth  in,  477 
Methane  in  excretions,  541 
Methemoglobin,  351 
Methyl  glucosides,  hydrolysis  of,  218 
glyoxaline,  66 
guanidine,  388,  395 
formula,  194 
tetany,  389 

oleate,  synthesis  of,  223 
orange,  271 
pyrrole,  355 

oi-Methyl-d-glucoside,  73 
0-Methyl-d-glucoside,  73- 
Methylacetate,  hydrolysis  of,  439,  475 
Milk,  clotting  of,  249 


624 


INDEX  OF  SUBJECTS 


Milk,  coagulation  of,  by  rennet,  213 

composition  of,  590 

formation  of,  in  mammary  glands, 
236 

guaiac  test  for,  362 

osmotic  pressure  of,  261 

in  vegetarian  diet,  585 
Millon's  reaction,  122,  123 
Mineral  acids  in  protein  coagulation,  122 

constituents  of  tissue  fluids,  268-271 

requirements  of  organism,  34 
Mistletoe,  inosite  in,  96 
Molecular  concentration,  estimation  of, 
260 

solutions,  first  use  of,  in  physiology, 

310 

Molisch  test,  58 

Mollusca,   artificial  parthenogenesis  in, 
449 

hemocyanin  in,  350 
Monoamino-dicarboxylic  acids,  132 
Monoamino-phosphatids,  114 
Monoglycerides,  107 
Monomolecular  chemical  reaction,  475 

logarithmic  formula,  251 
Mononucleotids,  178 

absorption  of,  231 
Monosaccharides,  53 

distribution    of,    in   living    tissues, 

69-72 

Monourates,  548 
Monstrosities,  469-470 
Moore's  test  for  sugar,  58 
Motility  in  Balanus  larva,  calcium  neces- 
sary for,  325 
Mucic  acid,  64 

formula,  404 
Mucilages,  vegetable,  82 
Mucins,  129 

glucosamin  in,  69 
Mucoids,  129 

glucosamin  in,  69 
Multirotation,  72 

Murex,  ammonium  purpurate  in,  175 
Murexide  test,  549 
Muscle  element,  diagram  of,  444 

fatigue,  products  of,  524 

plasma,  398 
Muscular  activity  in  infants,  592 

contraction,  391 

chemical  mechanics  of,  438 
reactions  underlying,  428 

exertion,  stimulation  of  metabolism  \ 
in,  589 

tissues,  glucose  consumption  by,  in 
children,  592 

work,  568 

carbon  dioxide  output  in,  539 
respiratory  quotient  in,  538 
tissue  glycogen  in,  391 
Mutarotation,   72 

Mutton  tallow,  assimilation  of,  235 
Mylius'  reaction,  103 
Myogen.  398 

fibrin,  398 


Myogenic  contractions  in  skeletal  mus- 
cle, 312 

Myqneural    junction    of    sympathetics, 
stimulation  of,  369 

Myosin,  398 
fibrin,  398 

Myricyl  alcohol,  112 

Myristic  acid,  410 

Myrosin,  89 

Myxedema,  382,  386,  486 


N 


NAPHTHO-RESORCINOL  reaction,  67 
Narcotics,  lipoid  theory  of,  293 
Naunyn  plan,  407 
Negative  phase  of  intravascular  clotting, 

343 

Nephritis,  cholesterol  deposition  in,  237 
globulin-albumin  ratio  in,  341 
urea  excretion  in,  542 
Nerve  cells,  chemical  changes  in,  428 
passage  of  impulse  in,  523 
centers,  fatigue  products  of,  524 
fibers,  rate  of  conduction  of,  523 
impulse,  effect  of  temperature  on, 

426 
Nervous  system,  growth  catalyzers  in 

517 

in  inanition,  501 
tissues,  514 

growth  relationships  of,  500 
Neuberg-Rauchwerger's    reaction,     100, 

104 

Neurine,  116,  196 
Neurokeratin,  127 
Neurons,  522 

in  cerebral  cortex,  stimulation  of,  by 

creatine,  195 

Neutral  fat  in  circulation,  234 
in  diabetic  blood,  406 
as  source  of  acetone  bodies,  409 
salts  in  protein  coagulation,  122 
sulphur,  560 

output  of,  after  taurine  admin- 
istration, 561 

Neutrality  of  tissue  fluids,  271 
Neutralizing  power  of  blood,  277 

of  saliva,  279 

Newborn  infant,  heat  production  by,  591 
Nicotinic  acid,  191,  192 
Ninhydrin  reaction,  123 
Nitriles,  65 
Nitrogenous  bases,  173-200 

derived  from  guanidine,  194 
from  hydrolysis  of  nucleic 

acid,  174-184 
from  phospholipins,  196 
in  internal  secretions,  197-200 
production  of,  by  bacteria  and 

fungi,  184 
metabolism  580 
poisons,  514  </ 
waste  products,  541-554 


INDEX  OF  SUBJECTS 


625 


Nitrous  acid,  action  of,  on  amino-acids, 

138 

Normal  diet,  582 

Nuclear  material,  synthesis  of,  462 
Nuclease,  178 
Nucleic  acids,  173-183,  463,  549 

glucosides  of,  in  decomposition 

products,  90 
in  growth,  494 
iminazole  ring  in,  66 
in  nucleoproteins,  128 
as  source  of  phosphorus  in  food- 
stuffs, 50 
structure  of,  178 
Nuclein,  129,  173 

iron  content  of,  43 
Nucleohistone,  128 
Nucleoproteins,  128,  173 

iron  content  of,  43 
a-Nucleoproteins,  179 
/8-Nucleoproteins,  173,  179 
Nucleosides,  90,  178,  455 
Nucleotids,  178-183 
Nutrient  level,  485,  487,  501,  502,  515, 

547 
Nutrients  in  growth,  484 

partition  of,  500 
Nutrition  of  children,  590 
of  plants,  320 


OBERMULLER'S  reaction,  99 
Obesity,  512 

cures,  385 

Octadecapeptide,  142 
Oils,  effect  of,  on  surface  tension,  284 
Old  age,  512 
Oleic  acid,  108 
Oligodynamic  action,  328 
Olive  oil,  111 

effect  of,  on  surface  tension  of 

water,  285 
emulsifying   action   of  alkalies 

on,  286 
One-sided  permeability,  289,  329 

in  kidneys  and  intestinal  epi- 
thelium, 291 
Ontogeny,  261 
Oocytin,  455 
Optical  activity,  54,  435 
Optimum  temperature  for  enzyme  action, 

213 

Orcin  reaction,  62 

Organs  of  generation,  chemical  correla- 
tion of,  376-381 
Ornithine,  562 

in  benzoic  acid  elimination  by  birds. 

558 

formula,  544 
Ornithuric  acid,  558 
Oryzenin,  126 
Osazones,  60 
crystals,  61 
40 


Osmometer,  258 

Starling's,  302 

Osmosis,  time  relations  of,  265 
Osmotic  pressure,  166 

in  assimilation  and  excretion,  39 
of  cell  contents,  263-268 
methods  of  estimating,  260 
of  protein  solutions,  302 

effect  of  salts  on,  303 
of  sea  water,  446 
of  tissue  fluids,  255-263 
Osteomalacia,  43 
Ovary,  377 
Ovomucoid,  457 

coagulation  and  precipitation  of,  168 
Ovovitellin,  129 
Ovulation,  378 
Oxalated  blood,  342 

plasma,  344 
Oxalic  acid,  541 
Oxidases,  206 

artificial,  596 
Oxidation  of  linseed  oil,  111 

spontaneous,    autocatalytic    nature 

of,  439' 
Oxidations  in  central  nervous    system, 

501 

in  living  tissues,  365 
in  sea  urchin  eggs,  461 
Oxidizing  enzymes,  215,  412-414 
Oxygen  consumption  by  different   ani- 
mals, 587 
in  eggs,  effect  of  fertilization  on. 

459 
temperature  coefficient  of, 

423 

Oxygenase,  413 
Oxyhemoglobin,  366 

crystals  from  various  animals,  358 
p-Oxyphenylacetic  acid,  558 
p-Oxyphenylpropionic  acid,  558 
Oxyproline,  135,  356 
Oxyproteic  acids,  562 


PALMITIC  acid,  108 

oxidation  of,  in  body,  409 
Pancreas,  autolysis  of,  177 

effect  of  extirpation  of,  401 
Pancreatic  juice,  200,  229,  232 

hydrogen  ion  concentration  of, 

275 
quantitative  laws  of  hydrolysis 

by,  251 
time   relations  of  flow    of,   in 

digestion,  372 
Papain,  214 
Parabamic  acid,  177 
Paracasein,  130,  345 

formation  of,  by  gastric  juice,  248 
/3-Parahy droxyphenyl-a-amino  propioni  c 

acid,  134 
Parahydroxyphenylethylamine,   186-187 


626 


INDEX  OF  SUBJECTS 


Parallel  reactions,  501 
Paramecia,  action  of  ultraviolet  light -on, 
433 

effect  of  cholesterol  on  multiplica- 
tion of,  505 

of  coagulating  agents  on,  295 
Paranuclein,  130,  333 

synthesis  of,  224 
Parathyroids,  387 
Parenchymatous  tissues,  509,  590 
in  old  age,  515 
in  young  animals,  592 
Parthenogenesis,  446 
Parthenogenetic  frog,  450 
Pavement  epithelium,  one-sided  perme- 
ability in,  290 
Pawpaw,  enzymes  of,  214 
Pectase,  84 
Pectic  acid,  84 
Pectin,  82,  84 
Pectinase,  85 
Pectoses,  84 
Pellagra,  193 
Pentpsans,  84,  231 

in  vegetables,  71 
Pentoses,  53,  62 

derivation  of,  in  tissues,  231 

distribution  of,  in  tissues,  70 

in  oocytin,  455 
Pentose-d-ribose,  173 
Pentosuria,  70 
Pepsin,  120,  151,  224 

action  of,  on  nucleoproteins,  173 
on  polypeptides,  143 
on  iron  containing  protein,  45 
on  protein-protamine  com- 
pounds, 170 

effect  of  reaction  on,  217 

in  gastric  digestion,  247 

time  relations  in  hydrolysis  by,  210 
Peptamines,  199 
Peptides,  142 

artificial,  hydrolysis  of,  220 

in  protein  hydrolysis,  143 
Peptone  plasma,  343 
Peptones,  130,  132 

absorption  of,  238 

in  blood  coagulation,  342-343 

effect  of,  on  bacterial  decarboxyliza- 
tion,  184 

in  gastric  digestion,  247,  372 
Permeability  of  bloodvessels  for  lymph, 
363 

of  cell  surfaces,  326 

of  cells  for  unbalanced  solutions,  323 

of  kidney  in  glycosuria,  399 

one-sided,  289 

relative,  265 

role  in  protein  synthesis  by  tissues, 
246 

of  tissue  cells,  342 
Peroxidase,  362,  413 

hemoglobin  as,  413 

in  luminescence,  415 
Peroxides,  412 


Persulphate  reaction,  199 
Pettenkofer's  reaction,  103 
Phagocytes,  514 

Phenol-water  system,  phases  of,  298 
Phenolsulphonphthalein,  275 
Phenolsulphuric  acid  in  urine,  556 
Phenylacetic  acid,  412 
Phenylalanine,  556 

in  alcaptonuria,  559 
formula,  134 

ultraviolet  spectrum,  351,  433 
!  jS-Phenyl-a-aminopropionic  acid,  134 
a-Phenylbromopropionyl  chloride,  141 
|  Phenylcyanate-glycylglycine,  140 
Phenylethylamine,  effect  of,   on  blood- 
pressure,  186 
Phenylglucuronic  acid,  67 
Phenylhydrazine,  59 
I  Phenylhydrazones,  59 
!  Phloridzin  glycosuria,  398-399 
effect  of  inosite  on,  96 
Phloroglucin  reaction,  62 
Phosphates  in  blood,  277 

neutralizing  power  of,  278 
Phosphatids,  113-118 
Phosphoglobulins,  343 
Phospholipins,  113-118,  129 
in  diabetic  blood,  406 
in  nuclear  synthesis,  462-469 
Phosphoproteins,  129 
Phosphorus    compounds,    effect    of,    on 

metabolism,  311 
excretion,  562 
in  foodstuffs,  50 
poisoning,  fat  infiltration  of  liver  in, 

286 
fibrinogen  content  of  blood  in, 

348 

lactic  acid  output  in,  398 
urea  output  in,  543 
Phosphotungstic  acid  reaction,  199 
I  Pholinus,  luminescence  of,  415 
Photochemical  reaction,  430 

temperature  coefficient  of,  428 
\  Photogenin,  414 

'  Photographic  spectrum  of  blood,  350 
Photophelein,  414 
!  Photosensitive  substances,  429 
j  Photosynthesis  of  carbohydrates,  434 

temperature  coefficient  of,  429 
Phototropism,  429 
1  Photuris,  luminescence  of,  415 
Phrenosin,  91,  118 
Phylloporphyrin,  356 
Physeter  macrocephalus,  biliary  concre- 
tions of,  101 
spermaceti  in,  1 12 

,  Physiologically  balanced  solutions,  318 
Phytase,  95 
Phytosterols,  97-100 
Picramic  acid,  195 
Picric  acid,  195,  548 
Pigments,  urinary,  562 
Pilocarpine,  373 
Pilocarpus  jaborandi,  373 


INDEX  OF  SUBJECTS                                       627 

Pineal  gland,  595  Protamine  sulphate  in  chloroform  emui- 

relation   of,  to    secondary  sex               sions,  288 

characters,  500  synthesis,  224 
Pineapple,  proteolytic  enzyme  in,  214        Protamines,  126,  136 

Pituitary  gland,  117  compounds  of,  with  proteins,  170 

in  growth,  494  molecular  weight  of,  171 

hypertrophy  in  pregnancy,  379,  rate  of  extraction  of,  by  acid,  535 

381  Proteases,  206 

inosite  in,  93  Protein  coagulation,  164,  169 

Pituitrin,  118,  199,  379,  500  complexes,  330 

Placenta,  378  in  blood  plasmas,  359 

Planaria,  reversion  of  growth  in,  486  dissociation  of,  171 

Plant  nucleic  acid,  173  compounds,  148-151 

Plants,  growth  of,  482  dehydration  of,  167 

curves  of,  474  intake  on  hard  work,  583 

Plasma,  335  ions,  157 

Plasmolysis,  263  jellies,  swelling  of,  304-308 

temporary,  265  metabolism  in  work,  394 

Plasmolyzing  agents,  204  molecule,  types  of  union,  148-151 

Plattner's  crystallized  bile,  102  requirement  of  children,  592 

Pneumonia,  341  in  dietary,  578 

Poiseuille's  law,  301  salts,  306 

Polar  seas,  density  of  population  of,  426                   in  blood,  277 

Polarized  light,  435  solutions,  conductivity  of,  296 

Polyneuritis,  193  osmotic  pressure  of,  302-304 

Polypeptide  structure  of  proteins,   151-  structure  in,  297 

157  SJ  Hirers,  491 

Polypeptides,  132,  142  synthesis,  138 

ultraviolet  spectrum  of,  433  localization  of,  in  organism,  245 

in  urine,  554  in  tissues,  243 

Polysaccharides,  53,  81-89  Proteins,  568 

Positive  phase,  in  intravascular  clotting,  acid  combining  capacity  of,  156 

343  aldehyde  groupings  of,  436 

Posterior  lobe  of  pituitary,  499,  500  amphoteric  character  of,  151 

active  principle  of,   117  biological  values  of,  58 

Potassium  in  diet,  35  in  blood  serum,  339 

myronate,  89  calorific  value  of,  569 

in  tissues,  329  classification  of,  124-125 

Potentiometric  method,  273  coagulation  of,  by  salts,  158-169 

Preadolescent  hyperpituitarisrn,  495  combination  of,  with  precipitating 

Precipitating  power  of  salts,  158  ion,  164 

Precipitation  of  proteins,  158  of  connective  tissue,  composition  of, 

reactions  of  proteins,  122  245 


Precipitins,  239,  331 
Pregnancy,  toxemia  of,  395 
Pressor  principles,  197 


digestion  of,  238-249 
•in  emulsification,  288 
in  formation  of  acid  secretions,  328 


Primary  proteose,  130  free  carboxyl-group  in,  153 

free  ammo-groups  in,  150  general  characteristics  of,  120-122 

Products  of  growth,  493  in  growth  and  maintenance,  490 

accumulation  of,  482  heat  units  of,  393 

effect  of  removal  of,  513  hydrolytic  decomposition  products 

Proline,  356  of,  155 

formula,  135  inhibition  of  cytolysis,  456 

in  prolamines,  127  isodynamic  values  of,  577 

Prolamines,  127,  136  molecular  weight  of,  303 

Propionic  acid,  108  precipitation  of,  by  salts,  158-169 

in  oxidation  of  fats,  411  respiratory  quotient  of,  538 

Propyl  alcohol,  solvate  formation  of,  166           in  sclerous  tissue,  516 

Prosecretin,  373  as  source  of  muscle  lactic  acid,  398 

Prosthetic  group  in  lecithoproteins,  129           specific  dynamic  action  of,  578 

in  nucleoproteins,  128,  173  standard  requirement  of,  579 

Protagon,  118  i  Proteolytic  enzymes,  effect  of  reaction 

hydrolysis  of,  196  on,  217 

Protamine  casemate,   antigenic  proper-  I  specificity  of,  220 

ties  of,  332  I  Proteoses,  130,  132 


628 


INDEX  OF  SUBJECTS 


Prot  coses,  anticoagulation  of  blood,  343 

in  blood,  339 

effect  of,  on  lymph  flow,  363 

in  gastric  digestion,  247,  372 

primary,  free  amino-groups  in,  150 

toxic,  560 
Prothrombin,  344 
Protoplasm,  emulsion  structure  of,  284 

properties  of,  255-282 

viscosity  of,  295 
Protoplasmic  streaming,  444 
in  cell  division,  467 

surface  in  cell  division,  466 

tissues,  583,  589 
Prunus  laurocerasus,  photosynthesis  in, 

429 

Pseudoglobulin,  339 
Pseudopodia,  445,  468 
Ptyalin,  82,  228 
Puberty  gland,  376 

Purgation  by  calcium  precipitants,  315 
Purine  bases,  173,  174,  548 
formulae,  176 

nucleotid,  182 
Purines,  enzymatic  oxidation  of,  412 

in  relation  to  vitamines,  489 
Putrescine,  187,562 
Pyrimidine  bases,  173,  174 

formula,  174 

nucleotid,  182 

Pyrogallol,  luminescence  of,  415 
Pyrrole  group,  47 

grouping,  essentiality  of,  in  diet,  488 
Pyrrolidine  carboxylic  acid,  135 
Pythagoreans,  595 


QUANTITATIVE  relations  in  enzyme  hy- 
drolysis, 207-213 
Quebracho  bark,  inosite  in,  96 
Quillaja,  glucosides  in,  90 
Quinol,  oxidation  by  enzymes,  414 


R 


RACE,  influence  of,  on  growth,  484-488 
Radial  symmetry  in  cell  structure,  292 
Radiation  of  heat  from  body  surface,  588 

from  infants  and  adults,  592 
Raffinose,  81 

Ranafusca,  rate  of  development  of,  423 
Rate  of  absorption  from  intestine,  252 
of  development,  temperature  coeffi- 
cient, 423 
Rats,  growth  curves  of,  474 

on  deficient  diet,  492 
Reaction,    influence    of,    on    enzymatic 

hydrolyses,  217 
of  tissues   regulation  of,  276 
Reactions  and  tests,  acetyl  number  of 

fats,  110 
acid  number  of  fats,  1 10 


Reactions  and  tests,  Acree's,  123  - 

for  adrenaline,  198,  199 

alkaloidal  reagents,  122 

alloxan,  175 

for  alloxantin,  199 

for  aminotyrosine,  199 

benzidine,  362 

biuret,  123 

Cipollina's,  68 

coagulation,  122 

color  reactions  for  proteins,  122 

for  dihydrophenols,  199 

Ehrlich's,  69 

Fehling's  sugar  estimation,  58 

ferric   chloride  for  acetoacetic 
acid,  405 

Folin  and  Macallum's  reagent, 
191,  199,  549 

Gallois',  96 

Ganassini's,  549 

guaiac  for  blood,  362 

Hammarsten's,  103 

Hehner  number  of  fats,  110 

Heller's,  122 

hemin,  362 

Hopkins-Cole,  122 

iodine,  for  adrenaline,  198 
number  of  fats,  1 10 

Jaffe's,  195,  548 

Liebermann-Burchard,  99 

Lifschiitz's,  100 

mercuric   chloride   for  adrena- 
line, 198 

Millon's,  122 

Molisch,  58 

Moore's,  58 

inurexide,  175,  549 

Mylius',  103 

naphtho-resorcinol,  67 

Neuberg-Rauchwerger's,  100 

ninhydrin,  123 

Obermuller's,  99 

orcin,  62 

persulphate-adrenaline  test,  199 

Pettenkofer's,  103 

phenyl  hydrazine,  59 

phloroglucin,  62 

phosphotungstic  acid,  199 

precipitation,  of  proteins,  .122 

Reichert-Meissl  number  of  fats, 
110 

Salkowski's,  99,  548 

saponification  value  of  fats,  110 

Scherer's,  96 

Schiff's,  99,  549 

Schweitzer's  reagent,  83 

secretin  tests,  200 

Seliwanoff's,  62 

Stoke's  reagent,  351 

uric  acid  test,  199 

Weidel's,  175 

Weyl's,  195,  548 

Wheeler  and  Johnson's,  175 

Wollaston's,  562 

xanthoproteic,  122 


INDEX  OF  SUBJECTS 


629 


Reactivation  of  enzymes,  215 
Rectal  feeding,  252 
Reduced  hematin,  355 

hemoglobin,  351 
Regeneration,  482,  513 

temperature  coefficient,  424 

of  tissue,  547 

Reichert-Meissl,  number  of  fats,  1 10 
Rejuvenescence,  503 


Saponin  as  cytolytic  agents,  451 

effect  of,  on  surface  tension,  207,  281 
Sapotoxin,  90 
Sarcolactic  acid,  397 
Sarcomere,  442 
Sarcostyle,  442 
Sarcous  element,  442 
Scarlet  R,  287 
Scherer's  reaction,  96 


Relationship,  chemical,  of  animals  and  |  Schiff's  reaction,  99,  549 


plants,    3 1 

of  man  to  primates,  331 
Relative  permeability,  265 

semipermeability,  289 
Rennet,  249 

action  of,  on  casein,  130 
Rennin,  206 

time  relations  in  milk  clotting,  210 
Reptilia,  descent  of  birds  from,  262 
Respiration,  stimulation  of,  368 
Respiratory  activities,   chemical   corre- 
lation of,  365-368 

center,  365 

chemical  coordination  of,  524 
influence  of  temperature  on,  422 

movements,  365 

quotient,  537-539,  573 
Reticulin,  127 
Reversible  coagulation,  131 
Reversion  of  growth,  486 
Rhamnose,  231 

Rhus  succedanea,  laccase  from,  412 
d-Ribose,  69,  231 

formula,  70 
Ricin,  130 
Ricinoleic  acid,  439 
Ritinus,  castor  oil  in,  130,  474 
Rigor  mortis,  398 
Ringer's  solution,  268,  319 
Rubber,  properties  of,  599 


S 


Schizothcerus  nuttali,  trypsin  from,  224 

Schutz-Borrissov  rule,  211,  251 

Schweitzer's  reagent,  83 

Scilla,  glucosides  in,  89 

Sclerenchyma,  509 

Scleroproteins,  127 

Sclerous  tissues,  589 

in  old  age,  515 

Scutellaria,  glucuronic  esters  in,  67 

Scyllite,  96 

Sea  urchin  eggs,  artificial  parthenogene- 
sis of,  446-449 
cell  division  of,  468 
effects  of  oocytin  on,  455 
membrane  formation  in,   450- 

462 

monstrosities,  469 
nucleosides  in,  90,  455 
synthesis  of  nuclear  material  in, 

463-^65 
water  glycosuria,  271 

a  physiologically  neutral  fluid, 

reaction  of,  281 

Sealing  wax,  flow  of,  under  pressure,  298 
Seaweed,  iodine  content  of,  50 
Secalin,  86 
Secondary  proteose,  130 

sexual  characters,  376,  500 
Secretin,  200,  373,  375 
Selective  absorption  in  intestine,     246 
252 

in  living  tissues,  38,  50,  328 
Selivanoff's  test,  62,  71 
Semipermeability,  256,  263,  264 
Senna,  inosite  in,  96 
Sensitization  of  eggs,  454 
Sensory  stimulation,  428,  530 
Sericin,  128 
Serine,  134 


SACCHARIC  acid,  174,  404 

Salicylic  acid,  374 

Saline  cathartics,  315 

Saliva,  neutralizing  power  of,  261 

osmotic  pressure  of,  261 

sulphocyanides  in,  562 
Salivary  diastase,  time  relations  in  action  j  Seromucoid,  339 

of,  210  Serum,  336 

Salkowski's  reaction,  99,  548  albumin,  339 

Salmine,  126,  153,  224  globulin,  157,  339 

arginine  content  of,  136  sickness,  240 

compound  with  edestin,  170  |  Sex,  influence  of,  on  growth,  484 

Salmon  spermatozoa,  protamines  in,  126  i  Sex-linked  inheritance,  348 
Salt  antagonism,  321  ^ 

glycosuria,  399 

requirement  of,  in  diet,  35 
Sandow,  diet  of,  583 
Saponaria,  glucosides  in,  90 
Saponification,  107 

value  of  fats,  110 
Saponin,  90,  452,  455,  457 


Silica  hydrogel,  structure  of,  298 
Silicon  in  foodstuffs,  50 
Silk  fibroin,  144 
Simple  proteins,  126 
Sinalbin,  89 
Sinapis  alba,  89 

nigra,  89 
Sinigrin,  89 


630 


INDEX  OF  SUBJECTS 


Sitosterol,  100 
Skatol,  556,  563 

formula,  186 
Skatoxyl,  67 

Skatoxyl-glucuronic  acid,  67 
Skeletal  muscles,  329 
Senescence,  512,  601 

in  tethelin,  517 
Senescent  atrophy,  513,  600 

loss  of  weight,  512 
Sepia,  melanins  in,  414 
Sleep,  530 

Smilax,  glucosides  in,  90 
Snake  venoms,  343 

Soaps,  action  of,  on  pancreatic  secretion, 
374 

in  digestion  of  fats,  233 

diminution  of  surface  tension,  284, 

467 

Sodium  butyrate,  membrane   formation 
in,  451 

cacodylate,  342 

casemate,  224 

viscosity  and  conductivity  of, 
297 

chloride,  action  of,  on  muscles,  311 
in  artificial  parthenogenesis,  447 
as  foodstuff,  34 

fluoride,  kidney  injury  by,  291 

glycocholate,  102 

nitroprusside  test  of,  195,  548 

salts,  action  of,  on  muscles,  312 

taurocholate,  102,  233 
Soil,  bacterial  flora  of,  600 
Solanins,  451 

Solanum,  glucosides  in,  90 
Soluble  chitin,  89 
Solvates,  165-166 
d-Sorbinose,  58 
Sorbitol,  63 
South  Australian  infants,  growth  of,  476 

German  infants,  growth  of,  487 
Specific  dynamic  action,  578,  587 

gravity  of  urine,  563 
Specificity  of  blood,  362 

oxidizing  enzymes  of,  412 
Speed  of  reaction  in  enzyme  synthesis, 

223 

Sperm  whale,  101 
Spermaceti,  112 
Sphingol,  116 
Sphingomyelin,  116 
Sphingosine,  91,  116,  197 
Spinach,  iron  content  of,  47 
Spirogyra,  264 
Spongin,  121,  128,  599 
Staircase  phenomenon,  438 
Standard  deviation,  380 
Staphylococcus  infection,  globulin-albu- 
min ratio  in,  339 
Starch,  82,  85  ' 

digestion  of,  228 

hydrolysis  of,  86 

photosynthesis  of,  437 

synthesis  of,  by  diastase,  223 


Starvation,  amino-acid  content  of  blood 

in,  244 
effect  of,  on  globulin-albumin  ratio, 

342 

loss  of  weight  in,  500 
metabolism  of,  587 
respiratory  quotient  in,  539 
Statistical  methods,  26 
Stearic  acid,  108 

oxidation  of,  in  body,  409 
Stercobilin,  563 
Stereoisomerism,  55-58 
Stimulators  of  metabolism,  589 
Stoke's  reagent,  351 
Stomach,  absorption  from,  247-250 
Storage  of  potential  energy,  434 
Strawberries,    hypersensitivity    to    pro- 
teins of,  240 

Streptococcus    infections,    globulin-albu- 
min ratio  in,  339 
Slrongylocentrolus    purpuratus,    artificial 

parthenogenesis  of,  446-449 
Strontium  chloride,  454 
Strophanthus,  glucosides  in,  89 
Structure  in  collodion  gel,  298 
in  egg-white,  298 
in  gelatin,  299 
in  protein  solutions,  297 
of  protoplasm  and  enzymatic  syn- 
thesis, 225 

Sturgeon  sperm,  sturine  in,  136 
Sturine,  136,  153 
Substrate,  203,  440 

exhaustion  in  autocatalysis,  481 
of  growth,  482,  488,  489,  493,  577 
Succus  entericus,  238,  375 
Sucrose,  76 

photosynthesis  of,  437 
Sudan  III,  287 

Sugar  craving  in  children,  592 
excretion  of,  in  diabetes,  402 
intolerance  in  hyperpituitarism,  494 
solutions,  viscosity  of,  296 
Sugars,  chemical  relationships  of,  63-67 

lactone  structure  of,  72 
Sulphanilic  acid,  198 
Sulphocyanides  in  urine  and  saliva,  562 
Sulphur,  role  of,  in  foodstuffs,  50 
metabolism  of,  559-562 
neutral,  560 
in  urine,  560 
Sulphuric  acid  in  saliva  of  carnivorous 

molluscs,  281 

Summation  of  stimuli,  438 
Superficial  tension,  466 
Suprarenal  gland,  197 

cholesterol  esters  of,  in  cortex," 

93 

effect  of  removal  of,  368 
Suprarenin,  197 
|  Surface  law,  587 

tension,  207,  284 

in  colloid  precipitation,  160 
changes  in  muscle  contraction, 
441 


INDEX  OF  SUBJECTS 


631 


Surface  tension  engine,  441 

lowering  of,  in  emulsification  of 

fats,  233 

temperature  effect  on,  418 
Swelling  capacity  of  gelatin,  306 
of  gelatin,  307 
of  living  tissues,  307 
maximum,  307 
phenomena  in  muscular  contraction, 

441 

of  protein  jellies,  304-308 
Symbiosis,  81 

Sympathomimetic  bases,  186 
Syneresis,  335,  342 
Synthesis  of  enzymes,  597 
of  foodstuffs,  597 
by  hydrolyzing  enzymes,  221-226 
of  proteins,  138 

localization  of,  in  organism,  245 
Synthesizing  enzyme  from  pepsin,  224 
Syphilis,  globulin-albumin  ratio  in,  340 


T ACH  YC AKDIA  in  hyperthyroidism,  386 

Taka-diastase,  215 

Tannic  acid  precipitates  in  normal  and 

CO  blood,  352 
Taurine,  103,  561 
Taurocarbamic  acid,  561 
Teleostomi,  osmotic  pressure  of  serum, 

262 
Temperature,  effect  of,  on  carbon  dioxide 

output,  539 
on  enzymes,  213 
on  equilibrium  constant,  353 
on  heat  production  in  infants, 

591 

on  life  processes,  417 
on  osmotic  pressure,  258 
optimum  for  enzyme  action,  213 
regulation  of,  in  body,  540 
Temperature-coefficient  of  chemical  reac- 
tions, 216,  417 
of  life  duration,  424 
phenomena,  419 
of  nerve  conduction,  523 
of  tissue  respiration,  423 
Tenebrio  molitor,  basal  metabolism   of, 

422 

Terpenes,  93 

Testes,  effect  of  removal  of,  376 
Tetany  in  parathyroidectomy,  387 
in  thymus  fed  amblystoma,  389 

/ethelin,  95,  117,  199,  500,  506,  510 
in  carcinoma,  511 
effect  of,  on  life  duration,  516 

of  split  products  of,  on  uterus, 

199 

Tetradecapeptide,  142 
Tetraglyc3Tlglycin,  hydrolysis  of,  by  tryp- 

sin,  151 

Tetranucleotid,  178 
Tetrapeptides,  142 


Thalassochelys  curetta,  osmotic  pressure 

of  serum,  262 
Theobromine,  176,  563 
Theophylline,  176 
/3-Thio-a-aminopropionic  acid,  134 
Thoracic  duct,  lymph  flow  in,  363 
Threshold  of  sensory  stimulation,  530 
Thrombin,  343,  344 

action  of,  on  fibririogen,  345 
chemical  nature  of,  349 
Thrombokinase,  344,  346 
Thromboplastic  action  of  kephalin,  345 
Thymine,  174,  175 
Thymus,  389 

autolysis  of,  177 
growth  functions  of,  494 
histone,    combination    with    hemo- 
globin, 170 
Thymus  nucleic  acid,  174,  179,  389 

structure  of,  183 
Thyreoglobulin,  384 
Thyroid,  effect  of,  on  metabolism,  382 
in  exogenous  metabolism,  581 
extract,  effect  of,  on  globulin-albu- 
min ratio,  342 
iodine  content  of,  50 
in  tissue  development,  486 
Thyroxin,  384 
Time  relations,  digestion  and  absorption, 

250-252 

enzyme  action,  209 
memory,  529 

voluntary  movement,  527 
Tissue  fats,  composition  of,  235 

fluids,  mineral  constituents  of,  268- 

271 

neutrality  of,  271 
osmotic  pressure  of,  255-260 
respiration,    temperature-coefficient 

of,  423 

Tissues,  biological  individuality  of,  330 
factors  determining  composition  of, 

246 

selective  action  of,  328 
Titratable  alkalinity  of  blood,  273 
Tolerance,  315,  600 
Toluene,  membrane  formation  by,  451 
Tonus    of    muscles,     creatihe    content 

affected  by,  195,  399 
relation  of,  to  energy  consump- 
tion, 392 

Total  metabolism,  factors  of,  589 
Toxemias,  globulin-albumin  ratio  in,  341 
Toxic  action  of  salts,  319 
Trachoma,  581 
Transudate,  362 
Triacetyl  glucose,  enzymatic  synthesis  of, 

223 

Triglycerides,  107 
Trigonelline,  191 
Triketohydrinenehydrate,  123 
Trimethylglycine,  189 
Trimethylhistidine,  190 
Triolein,  224 
Trioxyglutaric  acid,  64 


632 


INDEX  OF  SUBJECTS 


Tripalmitin,  107 
Tripeptides,  142 
Tritico-nucleic  acid,  178,  183 
Tropical  climate,  effects  of,  541 
Trypsinogen,  346-374 
Trypsin,  151,  184,  205-206,  215,  346,  374 
action  of,  on  polypeptides,  143 
effect  of  peptic  digestion  on  action 

of,  248 

of  reaction  on,  217 
mode  of  action  of,  22 
from  mollusc  liver,  224 
specificity  of,  220 
time  relations  in  action  of,  210 
Tryptophane,  effect  of  administration  of 

on  indican  output,  555 
essentiality  of,  in  diet,  242 

for  tissue  accretion,  490 
formula,  135 

reactions  given  by  thrombin,  349 
Tubularia  crocea,  temperature-coefficient 

of  life  duration,  424 
Twin  formation,  469-470 
Tyramine,  187,  514 

effect  of,  on  blood-pressure,  186 
Tyrosinase,  369,  413 
Tyrosine  in  alcaptonuria,  559 

detoxication  of  ultraviolet  light  by, 

433 
essentiality  of,  for  tissue  accretion, 

490 

formula,  134 
in  Millon's  reaction,  123 
putrefaction  products  of,  556 
relation  of,  to  adrenaline,  198 

to  tyramine,  187 
separation  of,  133 
as  source  of  tyramine  in  intestine, 

187 
ultraviolet  spectrum  of,  351,  433 


U 


ULTRAVIOLET  light,  inactivation  of  en- 
zymes, 207 

spectrum,  351,  429,  433 

toxicity  of,  433 
Unicellular  organisms,  515 
Uracil,  174,  175 
Uranium,    formaldehyde    synthesis    by, 

Urates,  solubility  of,  552 
Urea,  393 

in  artificial  parthenogenesis,  446 

blood  content  of,  337 

excretion  of,  in  phosphorus  poison- 
ing, 543 

formation  of,  244 

formula,  541 

origin  of,  543 

parathyroid  control  of  formation  of, 
387 

permeability  of  blood  corpuscles  for, 
267 


Urea,  production  of,  in  body,  542 
Uric  acid,  176,  412,  548 

elimination  of,  553 
excretion  of,  by  birds,  543 
reagent,  191,  199 
test  for,  199,  549 
Uricase,  550 
Uricolysis,  550 
Uricolytic  index,  551 
Uridine,  178 
Urinary  pigments,  562 
Urine,  acidity  of,  564 
in  diabetes,  405 
excretion  of,  by  kidney,  291 
freezing-point  of,  261 
normal  composition  of,  565 
properties  of,  563 
specific  gravity  of,  563 
Urobilin,  562 
Urobilinogen,  562 
Urochrome,  562 
Uroerythrin,  562 
Uterine  contractions,  379 
Uterus,  action  of  pituitary  extract  on, 

199 

creatine  content  of,  in  pregnancy, 
399 


VALENCY  of  ions  in  colloid  precipitation, 

158 
rule  in  precipitation  of  colloids.  160- 

164 

Valerianic  acid,  108 
Valine,  134 

Van  Slyke  method  of  amino-N  determi- 
nation, 144-146 
of   CO2    determination     in 

blood,  279 
Van't  Hoff's  solution,  318,  447,  463 
Variability,  380 
Vas  deferens,  376 
Vasomotor  theory  of  sleep,  530 
Vaucheria,  antagonistic  salt  effects  of,  320 
Vegetable  glue,  599 
mucilages,  82 
oils  in  diet,  489 

in  replacement  of  animal  fats, 

577 

proteases,  206 

proteins,  amino-acids  in,  584 
Vegetarianism,  583 
Velocity  of  constants,  485 
of  forgetting,  533 
of  hydrolysis,  201-218 
Vinyltrimethylammonium  hydroxide,  196 
Viscosity,  effect  of,  on  conductivity,  296 

of  temperature  on,  418 
of  proteins,  effect  of  ionization  on, 

296 

of  protoplasm,  295 
Vital  force,  574 
Vitamines,  189-193,  489 
Vitellin,  glutamic  acid  in,  136 


INDEX  OF  SUBJECTS 


033 


Vividiffusion,  243 

Voluntary  movement,  time  relations  of. 
527 

W 

WAR  breads,  83 
Wassermann  reaction,  340 
Waste  products,  537-566 

.carbonaceous,  537-541 
nitrogenous,  541-554 
Water  as  food,  34 

percentage  of,  in  embryonic  tissue, 


in  tissues,  255 

soluble  B,  489 
Wax  palm,  113 
Waxes,  93,  111-113 
Weidel  reaction,  175 
Werner's  theory  of  valency,  156 
Weyl's  reaction,  195,  548 
Wheeler  and  Johnson's  reaction,  175 
White  of  egg,  structure  of,  298 

mice,  growth  of,  473,  499,  512 
Wilhelmy's  law,  201 
Witch's  milk,  379 
Wollaston's  test,  562 


XANTHINE,  176,  177 

enzymatic  oxidation  of,  412 

formula,  177,  549 

oxidase,  549 
Xanthoproteic  reaction  for  proteins,  122. 

123 

Xylitol,  64 
Xylose,  64 


YEAST,  191 

antineuritic  substances  in,  193 

selective  action  of,  435 

in  synthesis  of-  foodstuffs,  597 

Yeast-nucleic  acid,  178,  181-183 


Z 

ZEIN,  127,  150 

amino-groups  lacking  in,  490 
dietary  deficiencies  of,  577 

i  Zymoids,  214 

)  Zymotic  diseases,  601 


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